Papaya mosaic virus compositions and uses thereof for stimulation of the innate immune response

ABSTRACT

The use of compositions comprising a papaya mosaic virus (PapMV) moiety for stimulation of the innate immune response is provided. The PapMV moiety may be papaya mosaic virus or PapMV virus-like particles (VLPs). The PapMV compositions stimulate a sufficiently strong innate immune response to provide protection against subsequent pathogen challenge or to treat an established infection. The use of PapMV compositions to protect a subject from potential infection by a pathogen, such as a viral pathogen, a bacterial pathogen or a fungal pathogen, and the use of PapMV compositions to treat an established infection, are also provided. The PapMV compositions be administered, for example, via intranasal or pulmonary routes to elicit effects within the mucosa and/or in the respiratory system.

FIELD OF THE INVENTION

The present invention relates to the field of immunomodulation and, inparticular, to the use of compositions comprising papaya mosaic virus(PapMV) or PapMV virus-like particles to stimulate the innate immuneresponse.

BACKGROUND OF THE INVENTION

The ability of papaya mosaic virus (PapMV) and PapMV virus-likeparticles (VLPs) to enhance the immunogenicity of antigens has beendescribed in the following patent and patent applications.

U.S. Pat. No. 7,641,896, Canadian Patent Application No. 2,434,000, andInternational Patent Application No. PCT/CA03/00985 (WO 2004/004761)describe the use of PapMV or VLPs derived from PapMV coat protein forpotentiating an immune response to an antigen in an animal. Theantigen(s) may be attached to the PapMV or VLP or they may beadministered in combination with the PapMV or VLP.

International Patent Application No. PCT/CA2007/002069 (WO 2008/058396)describes influenza vaccines based on PapMV and PapMV VLPs. The vaccinescomprise PapMV or a PapMV VLP and one or more influenza antigens, whichmay be attached to the PapMV or VLP or may be administered incombination with the PapMV or VLP.

International Patent Application No. PCT/CA2007/001904 (WO 2008/058369)describes immunogenic affinity-conjugated antigen systems based onPapMV. This application describes fusions of PapMV coat protein with aplurality of affinity peptides capable of binding an antigen ofinterest.

International Patent Application No. PCT/CA2008/000154 (WO 2008/089569)describes vaccines against S. typhi and other enterobacterial pathogensbased on PapMV. The vaccines comprise PapMV or a PapMV VLP and one ormore enterobacterial antigens, which may be attached to the PapMV or VLPor may be administered in combination with the PapMV or VLP.

International Patent Application No. PCT/CA2009/00636 (WO 2010/012069)describes multivalent vaccines that comprise a PapMV component and oneor more antigens, and their use to provide protection against aplurality of strains of a pathogen, or against more than one pathogen.The vaccines can optionally comprise a Salmonella spp. porin component.

Other publications have described the ability of PapMV VLPs to elicithumoral and cellular immune responses to antigens (Denis et al., 2007,Virology, 363: 59-68; Denis et al., 2008, Vaccine 26: 3395-403; Leclercet al., 2007, J Virol, 81: 1319-26; Lacasse et al., 2008, J. Virol, 82:785-94). PapMV virus particles isolated from plants have been shown toactivate the innate immune response and to be recognized by the immunesystem as a pathogen associated molecular pattern (PAMP), leading to theproposal that the immunogenicity and intrinsic adjuvant properties ofPapMV translate into the specific long-lasting antibody responseobserved when PapMV is co-administered with antigens (Acosta-Ramirez etal., 2008, Immunology, 124: 186-97).

Innate immunity is the first line of antibody-independent defenseagainst infections and, in many instances, can eliminate infectiousagents. The components of innate immunity recognize structures that arecharacteristic of microbial pathogens and are not present on mammaliancells. The principle effector cells of innate immunity are neutrophils,mononuclear phagocytes, and natural killer (NK) cells. Neutrophils andmacrophages express surface receptors that recognize microbes in theblood and tissues, and either stimulate phagocytosis (e.g., mannose oropsonin receptors) or activate phagocytes not involved in ingestion(e.g., Toll-like receptors, TLRs). The effector mechanisms of innateimmunity are often used to eliminate microbes, even in an adaptiveimmune response. Thus, the innate immune response can provide signalsthat function in concert with antigen to stimulate the proliferation anddifferentiation of antigen-specific (adaptive) T and B lymphocytes.

Stimulators of the innate immune response have been described. U.S.patent application Ser. No. 11/830,622 (Publication No. 2008/0170966)describes methods of attenuating respiratory infection by inhalation ofa microbial lysate that stimulates innate immunity. U.S. patentapplication Ser. No. 10/972,062 (Publication No. 2009/0318337) describesmethods of activating innate immunity by administering proteosome-basedimmunoactive compositions to a subject. U.S. patent application Ser. No.12/556,759 (Publication No. 2011/0105383) describes methods forstimulation of innate immune resistance to pathogens by administering arecombinant bacterial protein to a subject.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide uses of papaya mosaicvirus compositions for stimulation of the innate immune response. Inaccordance with one aspect of the invention, there is provided a use ofa composition comprising Papaya Mosaic Virus (PapMV) or PapMV virus-likeparticles (VLPs) to stimulate an innate immune response in a subject andthereby prevent, or decrease the severity of, a microbial infection inthe subject.

In accordance with another aspect of the invention, there is provided ause of a composition comprising Papaya Mosaic Virus (PapMV) or PapMVvirus-like particles (VLPs) in the manufacture of a medicament tostimulate an innate immune response in a subject and thereby prevent, ordecrease the severity of, a microbial infection in the subject.

In accordance with another aspect of the invention, there is provided ause of a composition comprising Papaya Mosaic Virus (PapMV) or PapMVvirus-like particles (VLPs) to protect a subject against infection witha pathogen, wherein the composition stimulates the innate immuneresponse in the subject.

In accordance with another aspect of the invention, there is provided ause of a composition comprising Papaya Mosaic Virus (PapMV) or PapMVvirus-like particles (VLPs) in the manufacture of a medicament toprotect a subject against infection with a pathogen, wherein thecomposition stimulates the innate immune response in the subject.

In accordance with another aspect of the invention, there is provided ause of a composition comprising Papaya Mosaic Virus (PapMV) or PapMVvirus-like particles (VLPs) to treat a chronic or recurrent microbialinfection in a subject.

In accordance with another aspect of the invention, there is provided ause of a composition comprising Papaya Mosaic Virus (PapMV) or PapMVvirus-like particles (VLPs) in the manufacture of a medicament to treata chronic or recurrent microbial infection in a subject.

In accordance with another aspect of the invention, there is provided amethod of stimulating an innate immune response in a subject comprisingadministering to the subject an effective amount of a compositioncomprising papaya mosaic virus (PapMV) or PapMV virus-like particles(VLPs), thereby preventing, or decreasing the severity of, a microbialinfection in the subject.

In accordance with another aspect of the invention, there is provided amethod of protecting a subject against microbial infection comprisingadministering to the subject an effective amount of a compositioncomprising papaya mosaic virus (PapMV) or PapMV virus-like particles(VLPs), wherein the composition stimulates the innate immune response inthe subject.

In accordance with another aspect of the invention, there is provided amethod of treating a chronic or recurrent infection comprisingadministering to a subject having a chronic microbial infection acomposition comprising Papaya Mosaic Virus (PapMV) or PapMV virus-likeparticles (VLPs).

In accordance with another aspect of the invention, there is provided akit comprising a container having contained therein a pharmaceuticalcomposition comprising papaya mosaic virus (PapMV) or PapMV virus-likeparticles (VLPs), the container adapted to deliver the pharmaceuticalcomposition by an intranasal, pulmonary or vaginal route.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent inthe following detailed description in which reference is made to theappended drawings.

FIG. 1 presents (A) the amino acid sequence of the wild-type PapMV coatprotein (SEQ ID NO:1) and (B) the nucleotide sequence of the wild-typePapMV coat protein (SEQ ID NO:2).

FIG. 2 presents (A) the amino acid sequence of the modified PapMV coatprotein CPAN5 (SEQ ID NO:3), and (B) the amino acid sequence of modifiedPapMV coat protein PapMV CPsm (SEQ ID NO:4).

FIG. 3 presents results demonstrating that PapMV VLPs induce ananti-viral response that controls influenza infection, (A) weight lossof Balb/C mice (10 per group) treated intranasally with 30 or 75 μg ofPapMV VLPs or control buffer (PBS) and challenged with 100 pfu ofinfluenza virus strain WSN/33, (B) symptoms developed in the mice duringinfection (Symptoms: 0, No symptoms. 1, Lightly spiked fur, slightlycurved back. 2, Spiked fur, curved back. 3, Spiked fur, curved back,difficulty in moving and mild dehydration. 4, Spiked fur, curved back,difficulty in moving, severe dehydration, closed eyes and ocularsecretion), and (C) survival rate of the infected mice.

FIG. 4 presents results demonstrating that PapMV VLPs induce ananti-viral response that controls influenza infection, (A) weight lossof Balb/C mice (10 per group) treated intranasally with 60 μg of PapMVVLPs or control buffer (PBS) and challenged with 200 pfu of influenzavirus strain WSN/33, (B) symptoms developed in the mice during infection(Symptoms: as for FIG. 3), and (C) survival rate of the infected mice.

FIG. 5 presents results demonstrating that PapMV VLPs induce ananti-viral response that controls influenza infection, (A) depicts theweight loss of Balb/C mice (10 per group) treated intranasally withPapMV VLPs containing ssRNA, 60 μg PapMV VLPs containing poly I:C, 3 μgssRNA, 3 μg poly I:C, 60 μg of PapMV CP monomers or control buffer (TrisHCl 10 mM pH 8) and challenged with 200 pfu of influenza virus strainWSN/33, (B) presents a summary of the symptoms developed in the miceduring infection (Symptoms: as for FIG. 3).

FIG. 6 presents graphs indicating the presence of IP-10 (A) and IL-9 (B)in bronchoalveolar lavage of Balb/C mice treated intranasally with PapMVVLPs (60 μg), Pam3CSK4 (15 μg) or control buffer (Tris HCl 10 mM pH 8).Each point corresponds to the level of cytokines detected in each mouse.Also shown is the amount of IP-10 or IL-9 present in nasopharyngeallavage (“LNP”) from the mice.

FIG. 7 presents graphs indicating the presence of (A) MIP-1α, (B)MIP-1β, (C) MIP-2, (D) KC, (E) TNF-α, (F) RANTES, (G) VEGF, (H) MCP-1,(I) IP-10, (J) IL-17, (K) IL-13, (L) IL-12 (p70), (M) IL-9, (N) IL-6,(O) IL-1α, (P) IL-1β, (Q) GM-CSF and (R) G-CSF in bronchoalveolar lavageof Balb/C mice treated intranasally with one or two treatments of PapMVVLPs (60 μg) or with control buffer (Tris HCl 10 mM pH 8). Each pointcorresponds to the level of cytokines detected in each mouse.

FIG. 8 presents graphs depicting compilation of (A) CD86 and (B) CD69expression in DCs, CD8⁺ T cells and B cells of C57BL/6, TLR7 knockout(KO), MYD88 KO and IRF5/7 KO mice 24 h after PapMV VLP ssRNA (100 ng) orPBS immunization. Results were analyzed by FACS and are presented as aratio of the Mean Fluorescence Intensity (MFI) of the analyzed sample onthe PBS sample.

FIG. 9 presents graphs depicting compilation of flow cytometry analysisof (A) CD69, (B) MHC-I and (C) CD86 expression 24 h after immunizationof C57BL/6 mice with PapMV PapMV VLP ssRNA with or without treatmentwith an anti-BST2 antibody. ***p<0.001, **p<0.01, *p<0.05, NS: notsignificant.

FIG. 10 presents graphs depicting (A) evaluation by ELISA of thekinetics of production of IFN-α in serum and (B) spleen of C57BL/6 micefollowing immunization with 100 μg PapMV VLP ssRNA, and (C) ELISAquantification of serum IFN-α in C57BL/6 and different knockout mice 6 hpost-immunization with 100 ng PapMV VLP ssRNA or PBS.

FIG. 11 presents graphs depicting a compilation of CD86, MHC-I and CD69expression in (A) B lymphocytes and (B) dendritic cells from spleens ofC57BL/6 and IFNAR KO mice 24 h after immunization with PapMV VLP ssRNAor PBS, and (C) quantification by ELISA of antibody against PapMV VLPssRNA in serum of C57BL/6 and IFNAR KO mice at different time pointsafter PapMV VLP ssRNA immunization.

FIG. 12 presents a graph depicting the viral kinetics of LCMV clone 13in blood of C57BL/6 mice treated with 100 μg PapMV VLP ssRNA (filledsquares) or PBS (open circles) 6 hours before infection with 2×10⁶ PFULCMV clone 13 (titers are expressed in PFU per milliliter of blood; LOD:limit of detection).

FIG. 13 presents graphs depicting the viral titers in (A) spleen, (B)kidney, (C) liver and (D) brain of C57BL/6 and TLR7 knockout (KO) mice15 days after infection with 2×10⁶ PFU LCMV clone 13; mice were treatedwith 100 μg PapMV VLP ssRNA, 100 μg R837 or PBS 6 hours before infection(titers are expressed in PFU per organ). LOD: limit of detection.

FIG. 14 presents graphs depicting the proportion of CD8⁺ T cellsproducing (A) IFN-γ, (B) TNF-α and (C) both cytokines after GP33restimulation of splenocytes isolated from mice immunized with 100 μgPapMV VLP ssRNA, 100 μg R837 or PBS 6 hours before infection with 2×10⁶pfu LCMV clone 13 and sacrificed 15 days post-infection; (D) amount ofTFN-γ and (E) amount of TNF-α produced by CD8⁺ T cells after GP33restimulation, (F) Mean Fluorescence Intensity (MFT) of PD-1 expressionin GP33 specific CD8⁺ T lymphocytes, and (G) percentage ofDbGP33⁺CD8⁺CD44⁺ in splenocytes.

FIG. 15 presents graphs depicting the viral titers in (A) spleen, (B)kidney, (C) brain and (D) liver of C57BL/6 mice 45 days after infectionwith 2×10⁶ PFU LCMV clone 13; mice were treated with 100 μg PapMV VLPssRNA or PBS 6 hours before infection (titers are expressed in PFU perorgan). LOD: limit of detection.

FIG. 16 presents a chart depicting flow cytometry analysis of CD86expression in human PBMCs (CD14⁺CD11b⁺ cell population) 18 h afterstimulation with PapMV VLP ssRNA (MFI: mean fluorescence intensity).

FIG. 17 presents a graph depicting survival of mice treated with 2 dosesof PapMV VLP ssRNA at 2-week intervals prior to challenge with asub-lethal dose of Streptococcus pneumoniae.

FIG. 18 presents graphs depicting viral load in mice chronicallyinfected with LCMV and treated i.v. once/day with 100 μg PapMV VLP ssRNA(A) at day 1, 2, 3, 4 and 5 post-infection, and (B) at day 6 and 7post-infection only (titers are expressed in PFU/mL).

FIG. 19 presents graphs depicting the viral titers in different organsof mice treated as described for FIG. 18 at day 15 (end of theexperiment).

FIG. 20 presents a graph illustrating weight loss in mice treated once(1×), twice (2×), 5 times (5×) and 10 times (10×) at 1-week intervalswith PapMV VLPs and challenged with the influenza WSN/33 virus 3 daysafter the last treatment.

FIG. 21 presents electron micrographs showing cells found inbroncho-alveolar lavage (BAL) from mice treated with (A) buffer and (B)PapMV VLPs (neutrophils are circled), and (C) a graph depicting numbersof neutrophils found in the BAL.

FIG. 22 presents graphs depicting the IgG and IgG2a titers measured inthe blood of mice immunized intranasally with PapMV VLPs combined withthe trivalent inactivated flu vaccine (TIV), (A) total IgG titers afterone immunization, (B) total IgG liters after two immunizations at 14-dayintervals, and (C) IgG2a titers measured after two immunizations.

FIG. 23 presents graphs depicting the antibody titers measured in miceimmunized as described for FIG. 22 after two immunizations, (A) IgAtiters in the broncho-alveolar lavage (BAL), (B) total IgG titers in theBAL, and (C) IgA in the faeces.

FIG. 24 presents a graph showing weight loss in mice immunized asdescribed for FIG. 22 and challenged at day 15 with 1LD₅₀ of theinfluenza WSN/33 virus; weight loss was followed over a 14 day period.

FIG. 25 presents electron micrographs of (A) PapMV VLPs self-assembledwith ssRNA. and (B) PapMV VLPs self-assembled with poly I:C (dsRNA).

FIG. 26 presents a graph demonstrating that PapMV VLPs interact withTLR-2 and CD14 in a human monocyte cell line (THP-1) and that thisinteraction is blocked with antibodies (Ac) to TLR-2 and CD 14.

FIG. 27 presents a flow chart outlining the steps for the preparation ofin vitro assembled PapMV VLPs containing ssRNA in accordance with oneembodiment of the invention (rCP=recombinant PapMV coat protein;SRT=synthetic RNA template; rVLP=recombinant VLP; Ec.prCP=E. colicontaining plasmid encoding rCP; Ec. pSRT=E. coli containing plasmidencoding SRT).

FIG. 28 presents the sequence of a synthetic RNA template (SRT) [SEQ IDNO:5] used in one embodiment of the process outlined in FIG. 27; all ATGcodons have been mutated for TAA stop codons (bold), the first 16nucleotides (underlined) are from the T7 transcription start sitelocated within the pBluescript expression vector and the PapMVnucleation site for rVLP assembly is boxed.

FIG. 29 presents the sequence of a synthetic RNA template (SRT) [SEQ IDNO:6] used in one embodiment of the process outlined in FIG. 27; thefirst G is the first nucleotide of the transcript; all ATG codons havebeen mutated for TAA stop codons (bold); the first 1500 nucleotides ofthe sequence are identical to SEQ ID NO:5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for compositions comprising a papayamosaic virus (PapMV) moiety (referred to herein as “PapMV compositions”)and their use for stimulation of the innate immune response. The PapMVmoiety comprised by the PapMV compositions may be papaya mosaic virus orPapMV virus-like particles (VLPs). While the ability of PapMV toactivate the innate immune response has been noted previously ascontributing to the adjuvant effect of PapMV (Acosta-Ramirez et al.,2008, ibid.), it is demonstrated herein that when administered alone,PapMV and PapMV VLPs unexpectedly are also capable of stimulating aninnate immune response that is sufficiently strong to result inprotective and/or therapeutic effects. Moreover, the PapMV compositionsare capable of stimulating an immune response at mucosal surfaces.

In certain embodiments, therefore, the present invention provides forthe use of PapMV compositions to stimulate a protective non-specificimmune response in an animal and thus provide protection againstsubsequent pathogen challenge. As demonstrated herein, the PapMVcompositions are capable of stimulating a rapid innate immune responseand can provide protection against pathogen infection for several days.In certain embodiments, the present invention provides for the use ofPapMV compositions to protect a subject from potential infection by apathogen that gains access to the body via mucosal membranes. Forexample, the PapMV composition may be administered alone in order tostimulate the innate immune response or may be administered incombination with one or more antigens and act as a mucosal adjuvant thatallows for the generation of an effective mucosal immune response. Inaccordance with certain embodiments of the invention, therefore, thePapMV compositions are administered via a mucosal route and elicit aprotective effect within the mucosa and/or in the respiratory system. Incertain embodiments of the invention, the pathogen is one or more of aviral pathogen, a bacterial pathogen or a fungal pathogen.

Certain embodiments of the invention provide for the use of PapMVcompositions to treat an established infection, for example, aninfection with a viral pathogen, a bacterial pathogen or a fungalpathogen. In some embodiments, PapMV compositions may be used to treatan infection at a mucosal surface, for example, in the lungs, intestinesor genitourinary system.

Some embodiments of the invention provide for the use of PapMVcompositions to decrease the viral load in a subject with a chronicviral infection and thus assist with management and/or clearance of theinfection. Combination therapies using PapMV compositions andconventional therapies for chronic infection are also provided in someembodiments. In some embodiments, such combination therapies may, forexample, result in one or more of an improved efficacy of theconventional therapy, a decrease in the dosage amount of theconventional therapy required to reach a predetermined endpoint, adecrease in the duration of treatment, a decrease in side-effectsassociated with the conventional therapy, or the like. Certainembodiments of the invention provide for the use of PapMV compositions,alone or in combination with a conventional therapy, to treat immuneexhaustion in a subject with a chronic infection.

Typically, the compositions comprise the PapMV or PapMV VLPs and asuitable carrier or diluent, but compositions consisting of just thePapMV or PapMV VLPs in a suitable form for administration to a subject(for example, freeze dried or lyophilized) remain an alternative optionin some embodiments. Compositions comprising mixtures of PapMV and PapMVVLPs are also contemplated in certain embodiments.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the term “about” refers to approximately a +/−10%variation from a given value. It is to be understood that such avariation is always included in any given value provided herein.

The use of the word “a” or “an” when used herein in conjunction with theterm “comprising” may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one” and “one or more than one.”

As used herein, the words “comprising” (and grammatical variationsthereof, such as “comprise” and “comprises”), “having” (and grammaticalvariations thereof, such as “have” and “has”), “including” (andgrammatical variations thereof, such as “includes” and “include”) or“containing” (and grammatical variations thereof, such as “contains” and“contain”) are inclusive or open-ended and do not exclude additional,unrecited elements or method steps.

“Naturally occurring,” as used herein, as applied to an object, refersto the fact that an object can be found in nature. For example, anorganism, or a polypeptide or polynucleotide sequence that is present inan organism that can be isolated from a source in nature and which hasnot been intentionally modified by man in the laboratory is naturallyoccurring.

The terms “attenuate,” “inhibit,” “prevent” and grammatical variationsthereof, as used herein, refer to a measurable decrease in a givenparameter or event.

The term “vaccine,” as used herein, refers to a composition capable ofproducing a beneficial immune response when administered to a subject.

The term “pathogen,” as used herein, refers to an organism capable ofcausing a disease or disorder in a host including, but not limited to,bacteria, viruses, protozoa, fungi and parasites.

The term “subject” or “patient” as used herein refers to an animal inneed of treatment.

The term “animal,” as used herein, refers to both human and non-humananimals, including, but not limited to, mammals, birds and fish, andencompasses domestic, farm, zoo, laboratory and wild animals, such as,for example, cows, pigs, horses, goats, sheep or other hoofed animals,dogs, cats, chickens, ducks, non-human primates, guinea pigs, rabbits,ferrets, rats, hamsters and mice.

Administration of PapMV compositions “in combination with” one or morefurther therapeutic agents is intended to include simultaneous(concurrent) administration and consecutive administration. Consecutiveadministration is intended to encompass various orders of administrationof the therapeutic agent(s) and the composition of the invention to thesubject with administration of the therapeutic agent(s) and thecomposition being separated by a defined time period that may be short(for example in the order of minutes) or extended (for example in theorder of days or weeks).

The term “substantially identical,” as used herein in relation to anucleic acid or amino acid sequence indicates that, when optimallyaligned, for example using the methods described below, the nucleic acidor amino acid sequence shares at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity with a defined secondnucleic acid or amino acid sequence (or “reference sequence”).“Substantial identity” may be used to refer to various types and lengthsof sequence, such as full-length sequence, functional domains, codingand/or regulatory sequences, promoters, and genomic sequences. Percentidentity between two amino acid or nucleic acid sequences can bedetermined in various ways that are within the skill of a worker in theart, for example, using publicly available computer software such asSmith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J MolBiol 147:195-7); “BestFit” (Smith and Waterman, Advances in AppliedMathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus™,Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure,Dayhof, M. O., Ed pp 353-358; BLAST program (Basic Local AlignmentSearch Tool (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215:403-10), and variations thereof including BLAST-2, BLAST-P, BLAST-N,BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, and Megalign (DNASTAR)software. In addition, those skilled in the art can determineappropriate parameters for measuring alignment, including algorithmsneeded to achieve maximal alignment over the length of the sequencesbeing compared. In general, for amino acid sequences, the length ofcomparison sequences will be at least 10 amino acids. One skilled in theart will understand that the actual length will depend on the overalllength of the sequences being compared and may be at least 20, at least30, at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 110, at least 120, at least 130, atleast 140, at least 150, or at least 200 amino acids, or it may be thefull-length of the amino acid sequence. For nucleic acids, the length ofcomparison sequences will generally be at least 25 nucleotides, but maybe at least 50, at least 100, at least 125, at least 150, at least 200,at least 250, at least 300, at least 350, at least 400, at least 450, atleast 500, at least 550, or at least 600 nucleotides, or it may be thefull-length of the nucleic acid sequence.

The terms “corresponding to” or “corresponds to” indicate that a nucleicacid sequence is identical to all or a portion of a reference nucleicacid sequence. In contradistinction, the term “complementary to” is usedherein to indicate that the nucleic acid sequence is identical to all ora portion of the complementary strand of a reference nucleic acidsequence. For illustration, the nucleic acid sequence “TATAC”corresponds to a reference sequence “TATAC” and is complementary to areference sequence “GTATA.” The terms “corresponding to” and“corresponds to” when used herein to cross-reference a DNA and RNAsequence indicate that the DNA sequence is identical to all of a portionof the reference RNA sequence (or vice versa), however, the DNA sequencewill contain thymine (T) residues at positions corresponding to uracil(U) residues in the RNA sequence. Thus, for illustration, the DNAsequence “TATAC” corresponds to an RNA reference sequence “UAUAC.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

PapMV Moiety

The PapMV moiety in accordance with the present invention may be eitherPapMV or PapMV VLPs. In certain embodiments of the invention, thecompositions comprising VLPs may also comprise minor amounts ofmultimerised PapMV coat protein in the form of discs.

PapMV is known in the art and can be obtained, for example, from theAmerican Type Culture Collection (ATCC) as ATCC No. PV204™.

PapMV VIPs are formed from recombinant PapMV coat proteins that havemultimerised and self-assembled to form a VLP. When assembled, each VLPcomprises a long helical array of coat protein subunits. The wild-typevirus comprises over 1200 coat protein subunits and is about 500 nm inlength. PapMV VLPs that are either shorter or longer than the wild-typevirus can still, however, be effective. In certain embodiments of thepresent invention, a VLP may comprise at least 40 coat protein subunits.In some embodiments, a VLP may comprise between about 40 and about 1600coat protein subunits. VLPs comprising a greater number of coat proteinsare also contemplated. In some embodiments, a VLP may be at least 40 nmin length. In some embodiments, a VLP may be between about 40 nm andabout 600 nm in length. Certain embodiments of the invention contemplateVLPs of greater than 600 nm in length.

The VLPs in accordance with the present invention can be prepared from aplurality of recombinant coat proteins having identical amino acidsequences, such that the final VLP when assembled comprises identicalcoat protein subunits, or the VLP can be prepared from a plurality ofrecombinant coat proteins having different amino acid sequences, suchthat the final VLP when assembled comprises variations in its coatprotein subunits.

The PapMV coat protein used to prepare the VLPs can be the entire PapMVcoat protein, or part thereof, or it can be a genetically modifiedversion of the wild-type PapMV coat protein, for example, comprising oneor more amino acid deletions, insertions, replacements and the like,provided that the coat protein retains the ability to self-assemble intoa VLP. The amino acid sequence of the wild-type PapMV coat (or capsid)protein is known in the art (see, Sit, et al., 1989, J. Gen. Virol.,70:2325-2331, and GenBank Accession No. NP_(—)044334.1) and is providedherein as SEQ ID NO:1 (see FIG. 1A). Variants of this sequence areknown, for example, the coat proteins of Mexican isolates of PapMVdescribed by Noa-Carrazana & Silva-Rosales (2001, Plant Science, 85:558)have 88% sequence identity with SEQ ID NO:1. The nucleotide sequence ofthe PapMV coat protein is also known in the art (see, Sit, et al.,ibid., and GenBank Accession No. NC_(—)001748 (nucleotides 5889-6536))and is provided herein as SEQ ID NO:2 (see FIG. 1B).

As noted above, the amino acid sequence of the PapMV coat protein neednot correspond precisely to the parental (wild-type) sequence, i.e. itmay be a “variant sequence.” For example, the PapMV coat protein may bemutagenized by substitution, insertion or deletion of one or more aminoacid residues so that the residue at that site does not correspond tothe parental (reference) sequence. One skilled in the art willappreciate, however, that such mutations will not be extensive and willnot dramatically affect the ability of the recombinant PapMV CP toassemble into VLPs. The ability of a variant version of the PapMV coatprotein to assemble into VLPs can be assessed, for example, by electronmicroscopy following standard techniques, such as the exemplary methodsdescribed in U.S. patent application Ser. No. 11/556,678.

Recombinant PapMV CPs prepared using fragments of the wild-type coatprotein that retain the ability to multimerise and assemble into a VLP(i.e. are “functional” fragments) are, therefore, also contemplated bythe present invention. For example, a fragment may comprise a deletionof one or more amino acids from the N-terminus, the C-terminus, or theinterior of the protein, or a combination thereof. In general,functional fragments are at least 100 amino acids in length. In oneembodiment of the present invention, functional fragments are at least150 amino acids, at least 160 amino acids, at least 170 amino acids, atleast 180 amino acids, and at least 190 amino acids in length. Deletionsmade at the N-terminus of the wild-type protein should generally deletefewer than 13 amino acids in order to retain the ability of the proteinto self-assemble.

In certain embodiments of the present invention, when a recombinant coatprotein comprises a variant sequence, the variant sequence is at leastabout 70% identical to the reference sequence. In some embodiments, thevariant sequence is at least about 75% identical to the referencesequence. In other embodiments, the variant sequence is at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 97% identical, and at least about 98% identical to thereference sequence. In certain embodiments, the reference amino acidsequence is SEQ ID NO:1 (FIG. 1A).

In certain embodiments of the present invention, the PapMV CP used toprepare the recombinant PapMV CP is a genetically modified (i.e.variant) version of the PapMV coat protein. In some embodiments, thePapMV coat protein has been genetically modified to delete amino acidsfrom the N- or C-terminus of the protein and/or to include one or moreamino acid substitutions. In some embodiments, the PapMV coat proteinhas been genetically modified to delete between about 1 and about 10amino acids from the N- or C-terminus of the protein, for examplebetween about 1 and about 5 amino acids.

In certain embodiments, the PapMV coat protein has been geneticallymodified to remove one of the two methionine codons that occur proximalto the N-terminus of the wild-type protein (i.e. at positions 1 and 6 ofSEQ ID NO:1) and can initiate translation. Removal of one of thetranslation initiation codons allows a homogeneous population ofproteins to be produced. The selected methionine codon can be removed,for example, by substituting one or more of the nucleotides that make upthe codon such that the codon codes for an amino acid other thanmethionine, or becomes a nonsense codon. Alternatively all or part ofthe codon, or the 5′ region of the nucleic acid encoding the proteinthat includes the selected codon, can be deleted. In some embodiments ofthe present invention, the PapMV coat protein has been geneticallymodified to delete between 1 and 5 amino acids from the N-terminus ofthe protein. In some embodiments, the genetically modified PapMV coatprotein has an amino acid sequence substantially identical to SEQ IDNO:3 (FIG. 2A) and may optionally comprise a histidine tag of up to 6histidine residues. In some embodiments, the PapMV coat protein has beengenetically modified to include additional amino acids (for examplebetween about 1 and about 8 amino acids) at the C-terminus that resultfrom the inclusion of one or more specific restriction enzyme sites intothe encoding nucleotide sequence. In some embodiments, the PapMV coatprotein has an amino acid sequence substantially identical to SEQ IDNO:4 (FIG. 2B) with or without the histidine tag.

When the recombinant PapMV coat protein is prepared using a variantPapMV CP sequence that contains one or more amino acid substitutions,these can be “conservative” substitutions or “non-conservative”substitutions. A conservative substitution involves the replacement ofone amino acid residue by another residue having similar side chainproperties. As is known in the art, the twenty naturally occurring aminoacids can be grouped according to the physicochemical properties oftheir side chains. Suitable groupings include alanine, valine, leucine,isoleucine, proline, methionine, phenylalanine and tryptophan(hydrophobic side chains); glycine, serine, threonine, cysteine,tyrosine, asparagine, and glutamine (polar, uncharged side chains);aspartic acid and glutamic acid (acidic side chains) and lysine,arginine and histidine (basic side chains). Another grouping of aminoacids is phenylalanine, tryptophan, and tyrosine (aromatic side chains).A conservative substitution involves the substitution of an amino acidwith another amino acid from the same group. A non-conservativesubstitution involves the replacement of one amino acid residue byanother residue having different side chain properties, for example,replacement of an acidic residue with a neutral or basic residue,replacement of a neutral residue with an acidic or basic residue,replacement of a hydrophobic residue with a hydrophilic residue, and thelike.

In certain embodiments of the present invention, the variant sequencecomprises one or more non-conservative substitutions. Replacement of oneamino acid with another having different properties may improve theproperties of the coat protein. For example, as previously described,mutation of residue 128 of the coat protein improves assembly of theprotein into VLPs (Tremblay et al. 2006, FEBS J. 273:14-25). In someembodiments of the present invention, therefore, the coat proteincomprises a mutation at residue 128 of the coat protein in which theglutamic acid residue at this position is substituted with a neutralresidue. In some embodiments, the glutamic acid residue at position 128is substituted with an alanine residue.

Substitution of the phenylalanine residue at position F13 of thewild-type PapMV coat protein with another hydrophobic residue has beenshown to result in a higher proportion of VLPs being formed when therecombinant protein is expressed than when the wild-type proteinsequence is used (Laliberté-Gagné, et al., 2008, FEBS J.,275:1474-1484). In the context of the present invention, the followingamino acid residues are considered to be hydrophobic residues suitablefor substitution at the F13 position: Ile, Trp, Leu, Val, Met and Tyr.In some embodiments of the invention, the coat protein comprises asubstitution of Phe at position 13 with Ile, Trp, Leu, Val, Met or Tyr.In some embodiments, the coat protein comprises a substitution of Phe atposition 13 with Leu or Tyr.

In certain embodiments, mutation at position F13 of the CP may becombined with a mutation at position E128, a deletion at the N-terminus,or a combination thereof.

Likewise, the nucleic acid sequence encoding the PapMV coat protein usedto prepare the recombinant PapMV CP need not correspond precisely to theparental reference sequence but may vary by virtue of the degeneracy ofthe genetic code and/or such that it encodes a variant amino acidsequence as described above. In certain embodiments of the presentinvention, therefore, the nucleic acid sequence encoding the variantcoat protein is at least about 70% identical to the reference sequence.In some embodiments, the nucleic acid sequence encoding the variant coatprotein is at least about 75% identical to the reference sequence. Inother embodiments, the nucleic acid sequence encoding the variant coatprotein is at least about 80%, at least about 85% or at least about 90%identical to the reference sequence. In certain embodiments, thereference nucleic acid sequence is SEQ ID NO:2 (FIG. 1B).

Preparation of PapMV and PapMV VLPs PapMV

PapMV is known in the art and can be obtained, for example, from theAmerican Type Culture Collection (ATCC) as ATCC No. PV-204™. The viruscan be maintained on, and purified from, host plants such as papaya(Carica papaya) and snapdragon (Antirrhinum majus) following standardprotocols (see, for example, Erickson, J. W. & Bancroft, J. B., 1978,Virology 90:36-46).

PapMV VLPs

PapMV VLPs can be prepared by standard techniques known in the art (see,for example, Tremblay et al. 2006, ibid., and International PatentApplication Nos. PCT/CA2007/002069, PCT/CA2008/000154 andPCT/CA2009/00636), as well as by in vitro assembly techniques asdescribed herein.

Recombinant PapMV coat proteins for the preparation of PapMV VLPs can bereadily prepared by standard genetic engineering techniques by theskilled worker provided with the sequence of the wild-type protein.Methods of genetically engineering proteins are well known in the art(see, for example, Ausubel et al. (1994 & updates) Current Protocols inMolecular Biology, John Wiley & Sons, New York), as is the sequence ofthe wild-type PapMV coat protein (see, for example, SEQ ID NOs: 1 and2).

For example, isolation and cloning of the nucleic acid sequence encodingthe wild-type protein can be achieved using standard techniques (see,for example, Ausubel et al., ibid). For example, the nucleic acidsequence can be obtained directly from the PapMV by extracting RNA bystandard techniques and then synthesizing cDNA from the RNA template(for example, by RT-PCR). PapMV can be purified from infected plantleaves that show mosaic symptoms by standard techniques.

The nucleic acid sequence encoding the coat protein is then inserteddirectly or after one or more subcloning steps into a suitableexpression vector. One skilled in the art will appreciate that theprecise vector used is not critical to the instant invention. Examplesof suitable vectors include, but are not limited to, plasmids,phagemids, cosmids, bacteriophage, baculoviruses, retroviruses or DNAviruses. The coat protein can then be expressed and purified asdescribed previously and below.

Alternatively, the nucleic acid sequence encoding the coat protein canbe further engineered to introduce one or more mutations, such as thosedescribed above, by standard in vitro site-directed mutagenesistechniques well-known in the art. Mutations can be introduced bydeletion, insertion, substitution, inversion, or a combination thereof,of one or more of the appropriate nucleotides making up the codingsequence. This can be achieved, for example, by PCR-based techniques forwhich primers are designed that incorporate one or more nucleotidemismatches, insertions or deletions. The presence of the mutation can beverified by a number of standard techniques, for example by restrictionanalysis or by DNA sequencing.

One of ordinary skill in the art will appreciate that the DNA encodingthe coat protein can be altered in various ways without affecting theactivity of the encoded protein. For example, variations in DNA sequencemay be used to optimize for codon preference in a host cell used toexpress the protein, or may contain other sequence changes thatfacilitate expression.

One skilled in the art will understand that the expression vector mayfurther include regulatory elements, such as transcriptional elements,required for efficient transcription of the DNA sequence encoding thecoat or fusion protein. Examples of regulatory elements that can beincorporated into the vector include, but are not limited to, promoters,enhancers, terminators, and polyadenylation signals. Certain embodimentsof the present invention, therefore, provide vectors comprisingregulatory element operatively linked to a nucleic acid sequenceencoding a genetically engineered coat protein. One skilled in the artwill appreciate that selection of suitable regulatory elements isdependent on the host cell chosen for expression of the geneticallyengineered coat protein and that such regulatory elements may be derivedfrom a variety of sources, including bacterial, fungal, viral, mammalianor insect genes.

In the context of the present invention, the expression vector mayadditionally contain heterologous nucleic acid sequences that facilitatethe purification of the expressed protein. Examples of such heterologousnucleic acid sequences include, but are not limited to, affinity tagssuch as metal-affinity tags, histidine tags, avidin/streptavidinencoding sequences, glutathione-S-transferase (GST) encoding sequencesand biotin encoding sequences. The amino acids encoded by theheterologous nucleic acid sequence can be removed from the expressedcoat protein prior to use according to methods known in the art.Alternatively, the amino acids corresponding to expression ofheterologous nucleic acid sequences can be retained on the coat proteinif they do not interfere with its subsequent assembly into VLPs.

In one embodiment of the present invention, the coat protein isexpressed as a histidine tagged protein. The histidine tag can belocated at the carboxyl terminus or the amino tenninus of the coatprotein.

The expression vector can be introduced into a suitable host cell ortissue by one of a variety of methods known in the art. Such methods canbe found generally described in Ausubel et al. (ibid.) and include, forexample, stable or transient transfection, lipofection, electroporation,and infection with recombinant viral vectors. One skilled in the artwill understand that selection of the appropriate host cell forexpression of the coat protein will be dependent upon the vector chosen.Examples of host cells include, but are not limited to, bacterial,yeast, insect, plant and mammalian cells. The precise host cell used isnot critical to the invention. The coat proteins can be produced in aprokaryotic host (e.g. E. coli, A. salmonicida or B. subtilis) or in aeukaryotic host (e.g. Saccharomyces or Pichia; mammalian cells, e.g.COS, NIH 3T3, CHO, BHK, 293 or HeLa cells; insect cells or plant cells).

If desired, the coat proteins can be purified from the host cells bystandard techniques known in the art (see, for example, in CurrentProtocols in Protein Science, ed. Coligan, J. E., et al., Wiley & Sons,New York, N.Y.) and sequenced by standard peptide sequencing techniquesusing either the intact protein or proteolytic fragments thereof toconfirm the identity of the protein.

The recombinant coat proteins of the present invention are capable ofself-assembly into VLPs. Assembly of the VLPs can take place, forexample, in the host cell expressing the coat protein (see for example,Tremblay, et al., 2006, FEBS J., 273:14-25), or it may take place invitro, as described in more detail below (see also, International patentapplication Ser. No. ______ “Virus-Like Particles and Process forPreparing Same,” Filed May 1, 2012, herein incorporated by reference inits entirety). In some embodiments, the VLPs comprise ssRNA. Forexample, VLPs that are prepared by expression of recombinant PapMV coatprotein in E. coli and self-assembly of the coat protein in the bacteriamay comprise bacterial ssRNA. Alternatively, for VLPs assembled invitro, ssRNA may be added to the coat protein preparation prior toself-assembly. The ssRNA may be, for example, synthetic ssRNA, anaturally occurring ssRNA, a modified naturally occurring ssRNA, afragment of a naturally occurring or synthetic ssRNA, or the like.

Typically, the ssRNA for in vitro assembly is at least about 100nucleotides in length and up to about 5000 nucleotides in length, forexample, at least about 250, 300, 350, 400, 450 or 500 nucleotides inlength and up to about 5000, 4500, 4000 or 3500 nucleotides in length.In certain embodiments, the ssRNA for in vitro assembly is between about500 and about 3000 nucleotides in length. In certain embodiments, thessRNA for in vitro assembly is designed such that it does not includeany ATG start codons in order to minimize the chances of in vivotranscription of the sequences, however, inclusion of ATG start codonsis not excluded as in vivo transcription remains unlikely as the ssRNAis not capped. In certain embodiments, the ssRNA for in vitro assemblyincludes about 100 nucleotides from the 5′-end of the native PapMV RNA,which correspond to the putative packaging signal. ssRNA that does notinclude the putative packaging signal can also be assembledsuccessfully. Non-limiting examples of ssRNA based on the PapMV genomethat may be used in this regard are provided in FIGS. 28 and 29 [SEQ IDNOs: 5 and 6].

The VLPs can be isolated from host cells by standard techniques, such asthose described in Tremblay, et al. (2006, FEBS J., 273:14-25) and inthe Examples. The VLPs can be further purified by standard techniques,such as chromatography, if desired to remove contaminating host cellproteins or other compounds, such as LPS.

When required, the VLPs can be separated from the other coat proteincomponents by, for example, ultracentrifugation or gel filtrationchromatography (for example, using Superdex G-200) to provide asubstantially pure VLP preparation. In this context, by “substantiallypure” it is meant that the preparation contains 70% or greater of VLPs,for example, 80% or 90% or greater.

In certain embodiments, PapMV VLPs comprise ssRNA and are prepared by invitro assembly with a ssRNA template. Exemplary ssRNA template is shownin FIGS. 28 and 29 [SEQ ID NOs: 5 and 6], but one skilled in the artwill appreciate that various other ssRNA would be suitable for thispurpose, as described above. An exemplary method for in vitro assemblyis shown in FIG. 28 and described in Example 18. It will be recognizedthat various alterations may be made to this method and still provideVLPs. For example, expression of the recombinant coat protein and ssRNAtemplate can be effected in other host cells, such as Pichia pastoris,and the assembly reaction may be conducted at temperatures ranging from2° C. to 37° C. with various ratios of protein:ssRNA. In general, aprotein:ssRNA ratio between about 1:1 and about 50:1 by weight may beused, for example, between about 5:1 and about 50:1, between about 5:1and about 40:1, or between about 10:1 and about 40:1 by weight.

Characteristics of Recombinant PapMV Coat Proteins

Recombinant coat proteins can be analysed for their ability toself-assemble into VLPs by standard techniques. For example, byvisualising the purified protein by electron microscopy (see, forexample, Tremblay, et al., 2006, FEBS J., 273:14-25). In addition,ultracentrifugation may be used to isolate VLPs as a pellet, whileleaving smaller aggregates (20-mers and less) in the supernatant, andcircular dichroism (CD) spectrophotometry may be used to compare thesecondary structure of the recombinant or modified proteins with the WTvirus (see, for example, Tremblay et al., ibid.).

Stability of the VLPs and of PapMV can be determined if desired bytechniques known in the art, for example, by SDS-PAGE and proteinase Kdegradation analyses. According to some embodiments of the presentinvention, the PapMV VLPs are stable at elevated temperatures and can bestored easily at room temperature.

Evaluation of Efficacy

The efficacy of the PapMV compositions in stimulating the innate immuneresponse and producing a protective or therapeutic effect can beevaluated by standard techniques known in the art. For example, forprotective effects, challenge studies can be conducted. Such studiesinvolve the inoculation of groups of test animals (such as mice) withthe PapMV composition by standard techniques. Control groups comprisingnon-inoculated animals and/or animals inoculated with a known stimulatorof the innate immune response, or other positive control, are set up inparallel. After an appropriate period of time post-vaccination, theanimals are challenged with the pathogen of interest. The animals aremonitored for development of other conditions associated with infectionincluding, for example, body temperature, weight, and the like. Incertain cases, for example when the antigen is from certain strains ofinfluenza or other pathogens associated with mortality, survival is alsoa suitable marker. The extent of infection can also be assessed, ifdesired, by measurement of viral titers using standard techniques aftersacrifice of the animal.

For therapeutic studies, similar methods can be employed using standardanimal models of infection and with the animals being treated with thePapMV composition at an appropriate time post-infection.

In addition, blood samples collected from the animals at various timeintervals, for example pre-inoculation, post-inoculation and/orpost-challenge, can be analyzed for cytokine and/or chemokine inductionif desired using standard tests known in the art.

Other standard techniques may also be employed to assess thecompositions, including, for example, evaluation of efficacy incombination with conventional prophylactic or therapeutic drugs orvaccines in various animal models of infection and disease known in theart.

Pharmaceutical Compositions and Administration

The present invention provides for pharmaceutical compositionscomprising the PapMV moiety and one or more pharmaceutically acceptablecarriers, diluents and/or excipients. If desired, other activeingredients may be included in the compositions, for example, additionalimmune stimulating compounds, standard therapeutics, vaccines or thelike.

The pharmaceutical compositions can be formulated for administration bya variety of routes. For example, the compositions can be formulated fororal, topical, rectal, nasal or parenteral administration or foradministration by inhalation or spray. The term parenteral as usedherein includes subcutaneous injections, intravenous, intramuscular,intrathecal, intrasternal injection or infusion techniques. Intranasaladministration to the subject includes administering the composition tothe mucous membranes of the nasal passage or nasal cavity of thesubject.

In some embodiments, the pharmaceutical compositions are formulated formucosal administration. Mucosal administration may include, for example,oral, intranasal, aerosol, rectal or vaginal administration. Thepreparations for mucosal administration include transdermal devices,aerosols, creams, lotions or powders pending on the mucosal site. Incertain embodiments, the pharmaceutical compositions are formulated forintranasal or pulmonary administration. In some embodiments, thepharmaceutical compositions are formulated for rectal or vaginaladministration.

The pharmaceutical compositions comprise an effective amount of thePapMV moiety. The effective amount for a given indication can beestimated initially, for example, in animal models, usually in rodents,rabbits, dogs, pigs or primates. The animal model may also be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in the animal to be treated,including humans. In certain embodiments of the present invention, theunit dose comprises between about 10 μg to about 10 mg of protein, forexample, between about 10 μg to about 5 mg of protein, or between about40 μg to about 2 mg of protein. One or more doses may be used toimmunise the subject, and these may be administered on the same day orover the course of several days or weeks.

Compositions formulated as aqueous suspensions may contain the PapMVmoiety in admixture with one or more suitable excipients, for example,with suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, hydroxypropyl-β-cyclodextrin, gum tragacanth andgum acacia; dispersing or wetting agents such as a naturally-occurringphosphatide, for example, lecithin, or condensation products of analkylene oxide with fatty acids, for example, polyoxyethyene stearate,or condensation products of ethylene oxide with long chain aliphaticalcohols, for example, hepta-decaethyleneoxycetanol, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand a hexitol for example, polyoxyethylene sorbitol monooleate, orcondensation products of ethylene oxide with partial esters derived fromfatty acids and hexitol anhydrides, for example, polyethylene sorbitanmonooleate. The aqueous suspensions may also contain one or morepreservatives, for example ethyl, or n-propyl p-hydroxy-benzoate, one ormore colouring agents, one or more flavouring agents or one or moresweetening agents, such as sucrose or saccharin.

In certain embodiments, the pharmaceutical compositions may beformulated as oily suspensions by suspending the PapMV moiety in avegetable oil, for example, arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example, beeswax, hardparaffin or cetyl alcohol. These compositions can be preserved by theaddition of an anti-oxidant such as ascorbic acid.

In certain embodiments, the pharmaceutical compositions may beformulated as a dispersible powder or granules, which can subsequentlybe used to prepare an aqueous suspension by the addition of water. Suchdispersible powders or granules provide the PapMV moiety in admixturewith one or more dispersing or wetting agents, suspending agents and/orpreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example, colouring agents, can also be included in thesecompositions.

Pharmaceutical compositions of the invention may also be formulated asoil-in-water emulsions in some embodiments. The oil phase can be avegetable oil, for example, olive oil or arachis oil, or a mineral oil,for example, liquid paraffin, or it may be a mixture of these oils.Suitable emulsifying agents for inclusion in these compositions includenaturally-occurring gums, for example, gum acacia or gum tragacanth;naturally-occurring phosphatides, for example, soy bean, lecithin; oresters or partial esters derived from fatty acids and hexitol,anhydrides, for example, sorbitan monoleate, and condensation productsof the said partial esters with ethylene oxide, for example,polyoxyethylene sorbitan monoleate.

In certain embodiments, the pharmaceutical compositions may beformulated as a sterile injectable aqueous or oleaginous suspensionaccording to methods known in the art and using suitable one or moredispersing or wetting agents and/or suspending agents, such as thosementioned above. The sterile injectable preparation can be a sterileinjectable solution or suspension in a non-toxic parentally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol.Acceptable vehicles and solvents that can be employed include, but arenot limited to, water, Ringer's solution, lactated Ringer's solution andisotonic sodium chloride solution. Other examples include, sterile,fixed oils, which are conventionally employed as a solvent or suspendingmedium, and a variety of bland fixed oils including, for example,synthetic mono- or diglycerides. Fatty acids such as oleic acid can alsobe used in the preparation of injectables.

Optionally the pharmaceutical compositions may contain preservativessuch as antimicrobial agents, anti-oxidants, chelating agents, and inertgases, and/or stabilizers such as a carbohydrate (e.g. sorbitol,mannitol, starch, sucrose, glucose, or dextran), a protein (e.g. albuminor casein), or a protein-containing agent (e.g. bovine serum or skimmedmilk) together with a suitable buffer (e.g. phosphate buffer). The pHand exact concentration of the various components of the composition maybe adjusted according to well-known parameters.

Sterile compositions can be prepared for example by incorporating thePapMV moiety in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile compositions, some exemplarymethods of preparation are vacuum-drying and freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Contemplated for use in certain embodiments of the invention are variousmechanical devices designed for pulmonary or intranasal delivery oftherapeutic products, including but not limited to, nebulizers, metereddose inhalers, powder inhalers and nasal spray devices, all of which arefamiliar to those skilled in the art.

Metered dose inhalers typically use a propellant gas and requireactuation during inspiration. Dry powder inhalers use breath-actuationof a mixed powder. Nebulizers produce aerosols from solutions, whilemetered dose inhalers, dry powder inhalers, and the like generate smallparticle aerosols.

Some specific examples of commercially available devices suitable forthe practice of this invention are the ULTRAVENT® nebulizer(Mallinckrodt, Inc., St. Louis, Mo.), the ACORN II® nebulizer (MarquestMedical Products, Englewood, Colo.), the MISTY-NEB® nebulizer(Allegiance, McGraw Park, Ill.), the AEROECLIPSE® nebulizer (TrudellMedical International, Canada), the Accuspray™ nasal spray device(Becton Dickinson), the Mucosal Atomization Device (MAD300) (Wolfe ToryMedical), the OptiNose device (OptiNose, Oslo, Norway), the Nektar DPIsystem (Nektar Therapeutics, Inc., San Carlos, Calif.), the AERxpulmonary drug delivery system (Aradigm Corporation, Hayward, Calif.),the Spiros® device (Dura Pharmaceuticals), and the Respimat® device(Boehringer Ingelheim).

All such devices require the use of formulations suitable for thedispensing of the PapMV moiety. Typically, each formulation is specificto the type of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, adjuvants and/orcarriers useful in therapy as would be understood by a worker skilled inthe art. Also, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.

Thus, in some embodiments, the invention provides for pharmaceuticalcompositions that are formulated for delivery via an intranasal orpulmonary route in, for example, lyophilized powder form, in anaerosolized liquid form, or in a gel form. These routes ofadministration can also allow for easy administration in the event ofthe need for mass distribution.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the PapMV moiety in an aqueousmedium at a suitable concentration, for example, about 0.01 mg to 25 mg,or about 0.1 mg to 10 mg, of protein per mL of solution. The formulationmay also include a buffer and a simple sugar (for example, for proteinstabilization and regulation of osmotic pressure), and/or human serumalbumin ranging in concentration from about 0.1 to about 10 mg/ml.Examples of buffers that may be used include, but are not limited to,sodium acetate, citrate and glycine. Typically, the buffer will have acomposition and molarity suitable to adjust the solution to a pH in therange of 3 to 9. Generally, buffer molarities of from 1 mM to 50 mM aresuitable for this purpose. Examples of excipients, usually in amountsranging from about 1% to about 90% by weight (for example, from about 1%to about 50% by weight, or about 5% to about 30% by weight) of theformulation include, but are not limited to, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; alditols, such as mannitol, xylitol,xylose, maltitol, lactitol, xylitol sorbitol (glucitol), sorbitose,pyranosyl sorbitol, myoinositol and the like; and glycine, CaCl₂,hydroxyectoine, ectoine, gelatin, di-myo-inositol phosphate (DIP),cyclic 2,3-diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP),β-mannosylglycerate (firoin), β-mannosylglyceramide (firoin A), prolinebetaine and/or derivatives, as well as combinations thereof.

The nebulizer formulation may also contain a surfactant to reduce orprevent surface induced aggregation of the composition components causedby atomization of the solution in forming the aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbitan fatty acidesters. Amounts will generally range between about 0.001% and about 4%by weight of the formulation. A non-limiting example of a surfactant forthis purpose is polyoxyethylene sorbitan monooleate.

In certain embodiments, the pharmaceutical compositions can be deliveredin powder form using, for example, a metered dose inhaler device. Thispowder may be produced by lyophilization and may also contain astabilizer such as human serum albumin (HSA). Additionally, one or moreof the following may be added as an excipient to the composition, ifnecessary, to enhance one or more features (for example, to facilitatedispersal of the powder from a device, to increase the shelf-life of thecomposition, or to improve the stability of the composition duringlyophilization): monosaccharides such as fructose, maltose, galactose,glucose, D-mannose, sorbose, and the like; disaccharides, such aslactose, sucrose, trehalose, cellobiose, and the like; polysaccharides,such as raffinose, melezitose, maltodextrins, dextrans, starches, andthe like; alditols, such as mannitol, xylitol, xylose, maltitol,lactitol, xylitol sorbitol (glucitol), sorbitose, pyranosyl sorbitol,myoinositol and the like; and glycine, CaCl₂, hydroxyectoine, ectoine,gelatin, di-myo-inositol phosphate (DIP), cyclic 2,3-diphosphoglycerate(cDPG), 1,1-di-glycerol phosphate (DGP), β-mannosylglycerate (firoin),β-mannosylglyceramide (firoin A), proline, betaine and/or derivatives aswell as combinations thereof. The amount added to the composition canrange from about 0.01% to 200% (w/w), for example, from about 1% to 50%(w/w), or from about 5% to 30% (w/w) of the protein present. Suchformulations are then lyophilized and milled to the desired particlesize. Typically, the particles of the powder have a median diameter lessthan about 50 μm, for example, between about 1.5 μm and 10 μm. The meanparticle diameter can be measured using conventional equipment, such asa Cascade Impactor (Andersen, Ga.).

The powder may be suspended in a propellant with the aid of asurfactant. The propellant may be one of a variety of conventionalmaterials employed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

In certain embodiments of the invention, the pharmaceutical compositionsare administered intranasally and the compositions are thereforeformulated as nasal gels, creams, pastes or ointments that provide amore sustained contact with the nasal mucosal surfaces. Theseformulations typically have a viscosity between about 10 and about250,000 centipoise (cps), for example, between about 2500 about 100,000cps, or between about 5,000 and 50,000 cps. Such formulations may bebased upon, for example, alkylcelluloses and/or other biocompatiblecarriers of high viscosity well known to the art. A non-limiting exampleof an alkylcellulose is methylcellulose, which can be included in asuitable concentration, for example, between about 5 mg and about 1000mg per 100 ml of carrier, or between about 25 mg and about mg per 100 mlof carrier. In certain embodiments, the carrier containing the PapMVmoiety may be soaked into a suitable substrate, for example a fabricmaterial, such as gauze, that can be applied to the nasal mucosalsurfaces to allow for penetration of the PapMV moiety into the mucosa.

In certain embodiments, gel formulations may also include a permeationenhancer (penetration enhancer). Permeation enhancers include, but arenot limited to, sulfoxides such as dim ethylsulfoxide anddecylmethylsulfoxide; surfactants such as sodium laurate, sodium laurylsulfate, cetyltrimethylammonium bromide, benzalkonium chloride,poloxamer (231, 182, 184), Tween (20, 40, 60, 80) and lecithin; the1-substituted azacycloheptan-2-oncs, particularly1-n-dodecylcyclazacycloheptan-2-one; fatty alcohols such as laurylalcohol, myristyl alcohol, oleyl alcohol and the like; fatty acids suchas lauric acid, oleic acid and valeric acid; fatty acid esters such asisopropyl myristate, isopropyl palmitate, methylpropionate, and ethyloleate; polyols and esters thereof such as propylene glycol, ethyleneglycol, glycerol, butanediol, polyethylene glycol, and polyethyleneglycol monolaurate, amides and other nitrogenous compounds such as urea,dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone,1-methyl-2-pyrrolidone, ethanolamine, diethanolamine andtriethanolamine, terpenes; alkanones, and organic acids, particularlysalicylic acid and salicylates, citric acid and succinic acid. Thepermeation enhancer may be present in an amount from about 0.1% to about30% w/w. The gel compositions may also include a buffering agent, forexample, carbonate buffers, citrate buffers, phosphate buffers, acetatebuffers, hydrochloric acid, lactic acid, tartaric acid, inorganic andorganic bases. The buffering agent may be present in a concentration ofabout 1 to about 10 weight percent, for example, about 2 to about 5weight percent, depending on the type of buffering agent(s) used, asknown by the one skilled in the art. Concentrations of the bufferingagent(s) may vary, however, and in some embodiments the buffering agentmay replace up to 100% of the water amount within the composition.

In certain embodiments of the invention, the pharmaceutical compositionsare formulated for rectal or vaginal administration and may be presentedas a suppository, which may be prepared by mixing the activeingredient(s) with one or more suitable non-irritating excipients orcarriers. Non-limiting examples of excipients or carriers include cocoabutter, polyethylene glycol, a suppository wax or salicylate and whichis solid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive ingredient(s). Formulations of the present invention which aresuitable for vaginal administration also include pessaries, tampons,creams, gels, pastes, foams or spray formulations containing suchcarriers as are known in the art to be appropriate.

Also encompassed by the present invention are pharmaceuticalcompositions comprising the PapMV moiety in combination withcommercially available vaccines, for example, compositions formulatedfor intranasal or pulmonary administration.

Other pharmaceutical compositions and methods of preparingpharmaceutical compositions are known in the art and are described, forexample, in “Remington: The Science and Practice of Pharmacy” (formerly“Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams& Wilkins, Philadelphia, Pa. (2000).

Methods and Uses

The present invention provides for the use of PapMV compositions tostimulate the innate immune response in a subject. The subject may be ahuman or a non-human animal. The compositions are useful, for example,in the treatment or prevention of infection, including chronicinfection.

Certain embodiments provide for administration of PapMV compositions toa subject to protect the subject from potential infection by a pathogen.In accordance with certain embodiments of the invention, the PapMVcompositions are administered to elicit a protective effect within themucosa and/or in the respiratory system. Administration via intranasalor pulmonary routes, for example, can be used to provide protectionagainst respiratory pathogens. Administration via vaginal routes, forexample, can be used to provide protection against vaginal pathogens.Intranasal administration may also be effective to provide protectionagainst vaginal pathogens (see, for example, Holmgren & Czerkinsky,2005, Nature Medicine, 11(4):S45-S53). Other routes of administrationare also contemplated.

The innate immune response stimulated by administration of PapMVcompositions is non-specific and thus is expected to provide protectionagainst or treatment of a broad range of pathogens. The effects arerapid and thus may find utility in situations that require moreimmediate protection than a traditional vaccine may provide. Inaddition, as the effects are non-specific, administration of PapMVcompositions could provide immediate protection while the identity ofthe pathogen is determined and an appropriate vaccine or other treatmentidentified.

Certain embodiments of the invention provide for the administration ofPapMV compositions to a subject as a preventative or pre-emptive measureto protect against infection with a pathogen. Such an approach isuseful, for example, in immunocompromised patients (such as patientswith AIDS, patients under chemotherapy or patients takingimmunosuppressive drugs), in pandemic or epidemic situations to provideinitial protection to the population prior to development/distributionof an appropriate vaccine, to protect workers such as rescue workers,doctors and nurses entering areas of potential infection, and insituations where there is a threat of, or an incidence of, abioterrorism attack.

As demonstrated herein, administration of PapMV compositions increasedproduction of cytokines and chemokines within about 6 hours or less ofadministration and protection against challenge lasted for several days.Accordingly, in certain embodiments, the present invention contemplatesthe prophylactic administration of PapMV compositions to a subjectbetween about 4 hours and about a week prior to predicted exposure to asuspected pathogen, for example, at least 4, 5, 6, 7, 8, 9, 10, 11 or 12hours prior to exposure and up to about 7, 6, 5 or 4 days prior toexposure. Various ranges between each of these upper and lower limitsare also contemplated in certain embodiments. For example, between about4, 5, 6, 7, 8, 9, 10, 11 or 12 hours and about 7 days, between about 4,5, 6, 7, 8, 9, 10, 11 or 12 hours and about 6 days, between 4, 5, 6, 7,8, 9, 10, 11 or 12 hours and about 5 days, between about 4, 5, 6, 7, 8,9, 10, 11 or 12 hours and about 4 days.

Certain embodiments provide for the administration of multiple doses ofthe PapMV compositions in order to prolong the protection period.Administration of the doses would take place at specified timeintervals, for example, doses could be spaced by a period of betweenabout 12 hours to about 10 days. Certain embodiments provide for theadministration of a first dose followed by at least one subsequent doseand up to about 10 subsequent doses at time intervals of at least about12, 24, 36, 48 or 72 hours and up to about 10, 9, 8 or 7 days, forexample, between about 24 hours and about 7 days.

In certain embodiments, PapMV compositions may be administered toprovide protection against potential infection with a bacterialpathogen. Bacterial pathogens include, for example, various species ofthe Bacillus, Yersinia, Franscisella, Haemophilus, Streptococcus,Staphylococcus, Pseudomonas, Mycobacterium, and Burkholderia genus ofbacteria. In certain embodiments, PapMV compositions may be administeredto provide protection against potential respiratory infection with abacterial pathogen. Non-limiting examples of relevant pathogenicbacterial species include, but are not limited to, Bacillus anthracis,Yersinia pestis, Francisella tularensis, Streptococcus pnemoniae,Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia,Corynebacterium diphtheriae, Legionella pneumophila, Mycoplasmapneumoniae, Chlamydophila pneumoniae, Mycobacterium tuberculosis,Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae,Escherichia coli, Coxiella burnetii, Clostridia spp. and Shigella spp.In certain embodiments of the invention, PapMV compositions may beadministered to provide protection against potential infection with abacteria associated with bacterial pneumonia, for example, one or moreof S. pneumoniae, S. aureus, H. influenzae, K. pneumoniae, P.aeruginosa, E. coli, M. catarrhalis, C. burnetii, M. pneumoniae, L.pneumoniae, C. pneumoniae and Y. pestis. In certain embodiments of theinvention, PapMV compositions may be administered to provide protectionagainst infection with a vaginal or intestinal bacterial pathogen, forexample, Shigella spp., Salmonella spp., E. coli or Chlamydiatrachomatis.

In certain embodiments, PapMV compositions may be administered toprovide protection against potential infection with a viral pathogen.Viral pathogens include, for example, viruses from the familyAdenoviradae; Arenaviridae (for example, Ippy virus and Lassa virus);Birnaviridae; Bunyaviridae; Caliciviridae; Coronaviridae; Filoviridae;Flaviviridae (for example, yellow fever virus, dengue fever virus andhepatitis C virus); Hepadnaviradae (for example, hepatitis B virus);Herpesviradae (for example, human herpes simplex virus 1);Orthomyxoviridae (for example, influenza virus A, B and C);Paramyxoviridae (for example, mumps virus, measles virus and respiratorysyncytial virus); Picornaviridae (for example, poliovirus and hepatitisA virus); Poxyiridae; Reoviridae; Retroviradae (for example, BLV-HTLVretrovirus, HIV-1, HIV-2, bovine immunodeficiency virus and felineimmunodeficiency virus); Rhabodoviridae (for example, rabies virus), andTogaviridae (for example, rubella virus). Non-limiting examples ofrelevant pathogenic viruses include, but are not limited to, variousstrains of the influenza virus, cytomegalovirus, various strains ofrespiratory syncytial virus (including human respiratory syncytial virusand specific animal strains), various strains of parainfluenza virus(including human parainfluenza virus and specific animal strains),coronavirus (including human coronavirus and SARS coronavirus),rhinovirus (including human rhinovirus), enterovirus (including humanenterovirus), adenovirus (including human adenovirus), bocavirus(including human bocavirus), metapneumovirus (including humanmetapneumovirus), dengue virus, various hepatitis viruses, humanimmunodeficiency virus (HIV), West Nile virus, rabies virus, humanpapilloma virus (HPV), Epstein Barr virus (EBV) and polyoma virus. Incertain embodiments of the invention, PapMV compositions may beadministered to provide protection against potential infection with aninfluenza virus, a flavivirus (such as dengue fever virus or yellowfever virus), a parainfluenza virus, human metapneumovirus, respiratorysyncytial virus, coronavirus (such as SARS coronavirus), a rhinovirus oran adenovirus.

In certain embodiments, PapMV compositions may be administered toprovide protection against potential infection with a fungal pathogen.Fungal pathogens include, for example, Histoplasma capsulatum,Coccidiodes immitis, Blastomyces dermatitidis, Cryptococcus neoformans,Aspergillus fumigatus, Candida albicans and Pneumocystis carinii.

In certain embodiments, PapMV compositions may be administered toprovide protection against a biological weapon/bioterrorism agent.Examples of bioterrorism agents include, Category A bioterrorism agentssuch as anthrax (Bacillus anthracia), botulism (Clostridium botulinumtoxin), plague (Yersinia pestis), smallpox (variola major), tularemia(Francisella tularensis) and viral hemorrhagic fevers (filoviruses, suchas Ebola and Marburg, and arenaviruses, such as Lassa and Machupo);Category B bioterrorism agents such as brucellosis (Brucella species),epsilon toxin of Clostridium perfringens, glanders (Burkholderiamallei), melioidosis (Burkholderia pseudomallei), psittacosis (Chlamydiapsittaci), Q fever (Coxiella burnetii), ricin toxin from Ricinuscommunis (castor beans), staphylococcal enterotoxin B, typhus fever(Rickettsia prowazekii) and viral encephalitis (alphaviruses, such asVenezuelan equine encephalitis, eastern equine encephalitis and westernequine encephalitis); and Category C bioterrorism agents that includeemerging infectious diseases such as Nipah virus and hantavirus.

In certain embodiments, PapMV compositions may be administered toanimals in competition settings as a pre-emptive measure to protectagainst infection, for example, horse races, dog shows, cat shows andthe like. Administration of PapMV compositions to livestock inepidemic/pandemic situations is also contemplated in certainembodiments. Examples of common animal pathogens include, but are notlimited to, Bordetella bronchiseptica (the most common causative agentof “kennel cough”), canine distemper virus, canine adenovirus (type 1 or2), canine parainfluenzavirus (CPI), canine influenza virus (CIV),canine reovirus (type 1, 2 or 3), canine herpes virus, felineherpesvirus, feline calicivirus (FCV), Chlamydophila [Chlamydia]psittaci, bovine respiratory syncytial virus (BRSV), Bovine ViralDiarrhea (BVD) virus, Parainfluenza Type 3 (P13) virus, Haemophilussomnus, Mannheimia (Pasteurella) haemolytica, Pasteurella multocida,African swine fever virus (ASFV), classical swine fever virus (CSFV),peste de petits ruminants virus (PPRV), Nairobi sheep disease virus(NSDV), Actinobacillus pleuronomiae, Mycoplasma hyopneumoniae, swineinfluenza virus, various strains of avian influenza virus, equinearteritis virus, equine herpesvirus, various strains of equine influenzavirus, Rhodococcus equi and Streptococcus equi (for example, subspeciesequi and zooepidemicus).

In certain embodiments, PapMV compositions may be used in combinationwith intra-nasal vaccines to augment and extend the effects of thesevaccines. For example, certain influenza vaccines have been developedfor instranasal administration. Vaccines against pneumococcal bacteriaand Yersinia pestis.

In certain embodiments, PapMV compositions are administered to stimulatethe mucosal immune response in general and thus improve protection todiseases or infections of the intestine, genitourinary tract, and othermucosal surfaces including the lung. The PapMV compositions can beadministered, for example, via intranasal or pulmonary routes orintravaginally. In certain embodiments, PapMV compositions areadministered in combination with one or more antigens to stimulate themucosal immune response. In this context, the PapMV compositions areacting as a mucosal adjuvant that augments the effects of the antigen(s)in order to generate an effective mucosal immune response. Certainembodiments of the invention, therefore, provide for the use of thePapMV compositions, alone or in combination with one or more antigen, toprovide protection against infection with a micro-organisms that gainaccess to the body via mucosal membranes. Examples of suchmicro-organisms include, but are not limited to, Helicobacter pylori,Vibrio cholerae, Escherchia coli, Shigella spp., Clostridium difficile,rotaviruses, calici viruses, Mycoplasma pneumoniae, influenza virus,Mycobacterium tuberculosis, respiratory syncytial virus, HIV, Chlamydiatrachomatis, Neisseria gonorrhoeae and herpes simplex virus.

In certain embodiments, PapMV compositions may be used to treat aninfection, for example, an infection with a viral pathogen, a bacterialpathogen or a fungal pathogen such as those described above. In someembodiments, PapMV compositions may be used to treat an infection at amucosal surface, for example, in the lungs, intestines or genitourinarysystem.

Some embodiments of the invention provide for the use of PapMVcompositions to decrease the viral load in a subject with a chronic,persistent or recurrent infection and thus assist with management and/orclearance of the infection. Examples of chronic infections include, butare not limited to, HIV, AIDS and hepatitis C virus (HCV) infections.Examples of persistent and/or recurrent infections include, but are notlimited to, hepatitis B virus (HBV) infections, herpes simplex virus(HSV) infections, tuberculosis (caused by Mycobacterium tuberculosisinfection) and lyme disease (caused by Borrelia burgdorferi infection).

Combination therapies using PapMV compositions and conventionaltherapies for chronic infection are also provided in some embodiments.For example, in certain embodiments, PapMV compositions may be used incombination with PEG-interferon, ribavirin or PEG-interferon/ribavirintreatment for HCV, or in combination with nucleoside reversetranscriptase inhibitors (NRTIs), protease inhibitors (PIs), highlyactive anti-retroviral therapy (HAART), fusion inhibitors ornon-nucleoside reverse transcriptase inhibitors (NNRTT) for HIV andAIDS. In some embodiments, such combination therapies may, for example,result in one or more of an improved efficacy of the conventionaltherapy, a decrease in the dosage amount of the conventional therapyrequired to reach a predetermined endpoint, a decrease in the durationof treatment, a decrease in side-effects associated with theconventional therapy, or the like.

Certain embodiments of the invention provide for the use of PapMVcompositions, alone or in combination with a conventional therapy, totreat immune exhaustion in a subject with a chronic, persistent orrecurrent infection.

In certain embodiments, PapMV compositions can be administered viapulmonary routes to lung cancer patients to stimulate the anti-tumouractivity of the innate immune response in the lungs.

Kits

The present invention additionally provides for kits comprising PapMVcompositions. In certain embodiments the kit is portable and may becarried on a person. The kit may optionally further include a pathogendetector. The kit may also optionally contain a gas or mechanicalpropellant for the PapMV compositions.

Individual components of the kit would be packaged in separatecontainers and, associated with such containers, can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale. The kit mayoptionally contain instructions or directions outlining the method ofuse or administration regimen for the PapMV composition.

The components of the kits may be packaged as solutions, pessaries or inpowdered or lyophilized form. When components of the kit are provided indried or lyophilised form, the kit can additionally contain a suitablesolvent for reconstitution of the dried or lyophilised components.Irrespective of the number or type of containers, the kits of theinvention also may comprise an instrument for assisting with theadministration of the composition to a patient. Such an instrument maybe an inhaler, nebulizer, nasal spray device, syringe, pipette, pessarydispenser or similar medically approved delivery vehicle. In certainembodiments, the container comprising the composition may itself be suchan instrument.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It will be understood that theseexamples are intended to describe illustrative embodiments of theinvention and are not intended to limit the scope of the invention inany way.

EXAMPLES Example 1 Induction of an Antiviral Response in Mice byAdministration of PapMV VLPs #1

PapMV VLPs were prepared by expression of the PapMV coat protein andself-assembly in E. coli cells as previously described (Tremblay et al.,2006, FEBS J., 273:14-25) using PapMV coat proteins having a sequence asset forth in SEQ ID NO:4 (FIG. 2B). Balb/C mice (10 per group) weretreated twice at 7-day intervals with either 30 or 75 μg (50 μL volume)of PapMV VLPs administered by the intranasal route followed by anintranasal challenge with 100 pfu (plaque forming unit) of influenzavirus strain WSN/33 three days after the second treatment. Thedevelopment of infection was followed for 14 days. The weight of theanimal was measured once per day during these 14 days. Animals thatshowed more that 20% weight loss were sacrificed. The control group wastreated with PBS (saline buffer).

The results are shown in FIG. 3A-C. Both groups treated with the PapMVVLPs did not show signs of infection and continued to gain weightnormally (FIGS. 3A & B). In contrast, the group treated with PBS showedsigns of infection and had lost more than 12% of their weight at day 8after challenge (FIGS. 3A & B). PapMV VLPs improved the capacity of theanimals to fight influenza infection. All mice treated with the PapMVVLPs survived to the end of the experiment, whereas 30% of the controlgroup did not survive beyond day 8 after challenge (FIG. 3C).

Example 2 Duration of Protection to Viral Challenge Induced by PapMVVLPs

A similar experiment to that described in Example 1 was performed. ThePapMV VLPs were prepared by the same method and the same schedule oftreatments was employed, but using a different dose of PapMV VLPs (60μg). The challenge with influenza virus strain WSN/33 (200 pfu) wasperformed at days 10, 12, 14 and 17 (i.e. 3, 5, 7 and 10 days afterPapMV treatment) and symptoms were monitored for 14 days afterchallenge.

The results are shown in FIGS. 4A-C and show that PapMV VLPs providedprotection for up to 5 days after the last treatment. Protection wasevidenced by decreased weight losses (FIG. 4A) and lesser symptoms (FIG.4B) compared to control animals, as well as 100% survival (FIG. 4C), inthose animals challenged either 3 or 5 days after the last treatmentwith PapMV VLPs. The induced protection however faded by day 7, with aless than 50% survival rate observed in mice challenged at 7 or 10 daysafter the last treatment with PapMV VLPs, a survival rate comparablewith the control group treated with PBS.

The protection induced by the treatment with PapMV VLPs thus appears topersist for a period of about 5 days, a period that is consistent with anon-specific and non-persistent induction of the innate immune response.

Example 3 Induction of an Antiviral Response in Mice by AdministrationOf PapMV VLPs Containing Synthetic ssRNA

Polyinosinic-polycytidylic acid (poly I:C; dsRNA), a well knownToll-like receptor 3 (TLR-3) ligand, has been shown to be an inducer ofthe innate immune response in lungs through induction of the secretionof pro-inflammatory cytokines such as IL-6, CXCL10, JE, KC, mGCSF, CCL3,CCL5, and TNF (Stowell et al., 2009, Respir. Res., 10:43). TLR-7 is alsoknown to activate the innate immune response through the binding ofligands such as ssRNA and R837 (a guanosine analogue).

In an attempt to increase the capacity of the PapMV VLPs to elicit aninnate immune response and the development of an antiviral response,PapMV VLPs containing either poly I:C dsRNA or ssRNA were prepared bythe method described in Example 17. PapMV coat protein was assembled invitro with either poly I:C (dsRNA; InvivoGen, San Diego, Calif.) orssRNA to produce VLPs comprising the respective RNAs. The ssRNA wasprepared in vitro using the Promega T7 Ribomax Express large scale RNAproduction system (Promega, Madison, Wis.).

The assembled VLPs were examined by electron microscopy and observed tobe similar to VLPs prepared by the method described in Tremblay et al.(2006, FEBS 1, 273:14-25) (see FIG. 25A: PapMV VLPs containing ssRNA andFIG. 25B: PapMV VLPs containing poly I:C).

The efficacy of the two types of VLPs in inducing protection againstchallenge with influenza virus was evaluated. Balb/C mice (10 per group)were treated with 60 μg of PapMV VLPs containing ssRNA (“PapMV VLPssRNA”), PapMV VLPs containing poly I:C (“PapMV VLP poly I:C”) or withan equivalent amount of RNA (i.e. 3 μg of either poly I:C or ssRNA).Control mice were treated with 60 μg of PapMV coat protein (CP) monomers(without RNA) or with control buffer (10 mM Tris-HCl pH 8). Mice weretreated as described in Example 2 (i.e. intranasally twice at 7 dayintervals) and challenged 3 days after the last treatment with 200 pfuof influenza virus strain WSN/33. The weight, symptoms and survival ofthe animals were measured once per day during the following 14 days.Animals that showed more that 20% weight loss were sacrificed.

The results are shown in FIG. 5A-B. Mice treated with PapMV VLP ssRNAshowed the best performance of the treated groups. Specifically, micetreated with PapMV VLP ssRNA did not lose any significant amount ofweight (FIG. 5A) and showed very few, if any, symptoms (FIG. 5B). Thegroups treated with either PapMV VLP poly I:C or poly I:C alone showedpartial protection to the challenge with decreased weight losses (FIG.5A) and symptoms (FIG. 5B) as compared to the control group. Treatmentwith the PapMV CP monomers did not provide any protection with theamount of weight loss (FIG. 5A) and symptoms (FIG. 5B) observed in micetreated with the monomers being similar to that observed in mice treatedwith the PBS control. Subsequent analysis of the PapMV VLP poly I:Csuggested that these VLPs are not as stable as the PapMV VLP ssRNA,which may account for their poorer performance.

Example 4 Induction of Cytokines in Mice by Administration of PapMV VLPs#1

To elucidate the mechanisms induced by the PapMV VLP in the lungs, mice(5 per group) were treated following the same protocol as described inExamples 1 to 3 above (i.e. 2 treatments intranasally at 7 dayintervals) with 60 μg PapMV VLPs containing ssRNA, 15 μg of PamCSK4 (aTLR-2 ligand and non-inducer of IFN type 1) (Cedarlane, Burlington, ON)or with the control buffer (10 mM Tris HCl pH8). Broncho-alveolar lavage(BAL) was performed 24 hours after the second treatment and screened forthe presence of cytokines using Luminex technology (Milliplex Mousecytokine premixed 32-plex immunoassay kit; Millipore).

Two major cytokines, interleukin-9 (IL-9) and interferon-γ-inducedprotein 10 kDa (IP-10), were induced by treatment with PapMV VLPs orPamCSK4 (FIGS. 6A & B). IL-9 is a cytokine secreted by CD4+ Tlymphocytes that promotes T-cell proliferation and inhibition ofapoptosis. IP-10 appears as a result of the secretion of IFN-γ and playsan important role in recruitment of T-lymphocytes, dendritic cells, NKcells and macrophages at the site of stimulation. The induction of bothcytokines by PamCSK4 (which is a known a TLR-2 ligand and pathogenassociated molecular patterns (DAMP) molecule) and PapMV VLPs suggeststhat the VLPs may also be PAMPs.

Example 5 Induction Of Cytokines in Mice by Administration of PapMV VLPs#2

A similar experiment to that described in Example 4 was conducted exceptthat the BAL was performed 6 hours after treatment, and the treatmentswere either 1 or 2 administrations at 7 day intervals. As before, 60 μgof PapMV VLPs containing ssRNA were used in the experiment. Luminex (32cytokines detection kit) was used to screen for cytokine productionearly after treatment.

The results are shown in FIGS. 7A-R and demonstrate that 2 treatmentswith PapMV VLPs were more efficient than one treatment in inducingcytokines and chemokines in mice. In addition, a wider variety ofcytokines and chemokines were detected at 6 hours after treatment than24 hours after treatment (compare FIGS. 6 and 7).

MIP-1α, MIP-1β, MIP-2, mKC, TNF-α and MCP-1 were found to be veryabundant (FIGS. 7A-E and H) in BAL from mice treated with PapMV VLPs.These cytokines and chemokines activate human granulocytes (neutrophils,eosinophils and basophils) which can lead to acute neutrophilicinflammation. They also induce the synthesis and release of otherpro-inflammatory cytokines such as TNF-α, IL-6 and IL-1α/β fromfibroblasts and macrophages (Maurer and von Stebut, 2004, TheInternational Journal of Biochemistry & Cell Biology. 36: 1882-1886),which were also shown to be induced by PapMV VLPs (see FIGS. 7E, N, Oand P respectively). MIP-1 proteins can also promote health by inducinginflammatory responses against infectious pathogens such as viruses,including influenza virus (Menten et al., 2002, Cytokine Growth FactorReviews, 13: 455-481) and parasites (Aliberti et al., 2000, NatureImmunology, 1: 83-87), which is consistent with the results shown in thepreceding Examples.

IL-6 was also observed to be secreted in response to administration ofPapMV VLPs (FIG. 7N). Interestingly, IL-6 secretion was showed to berequired for resistance to infection by the bacteria Pneumococcuspneumoniae (van der Poll et al., 1997, J. Infect Dis., 176 (2): 439-44).

IP-10 was strongly induced by the treatment with PapMV VLPs (FIG. 7I).IP-10 is a chemotactic chemokine that favours the recruitment of T cellsat inflammatory sites and also favours proliferation and activation ofnatural killer cells (NK cells).

Interleukin 17 was also induced by the treatment with PapMV VLPs (FIG.7J). IL-17 is a cytokine that acts by increasing chemokine production invarious tissues to recruit monocytes and neutrophils to the site ofinflammation, similar to Interferon gamma. IL-17 is produced by T helpercells and is also a proinflammatory cytokinc that responds to theinvasion of the immune system by extracellular pathogens. IL-17coordinates local tissue inflammation through the upregulation ofproinflammatory cytokines and chemokines such as IL-6. granulocytecolony-stimulating factor, TNFα, IL-1, KC, MCP-1 and MIP-2 (Zepp et al.,2011, Trends Immunol. April 12. [Epub ahead of print]), which were alsoshown to be induced by PapMV VLP treatment.

PapMV VLP treatment (FIGS. 7Q & R) also induced G-CSF and GM-CSF, whichare known to stimulate stem cells to produce granulocytes (neutrophils,eosinophils and basophils) and monocytes. Monocytes exit the circulationand migrate into tissue, whereupon they mature into macrophages. Thus,G-CSF and GM-CSF are part of the inflammatory cascade by whichactivation of a small number of macrophages can rapidly lead to anincrease in their numbers, a process crucial for fighting infection(Metcalf, 2010, Nature Reviews Cancer, 20: 425-434).

The results described in Examples 4 and 5 demonstrate that the treatmentof mice with PapMV VLPs induces a strong and general inflammatoryresponse as showed by the profile of cytokines and chemokines that aresecreted by the immune cells. The levels of cytokines and chemokineswere maximal at 6 hours after treatment and decreased significantly 24hours after treatment. It is likely that the inflammatory cytokines andchemokines induced the migration of immune cells and granulocytes andthus are responsible for the anti-viral state of the animal for morethan 5 days. The induced cytokines can also lead to secretion of IFNtype 1 that in turn is also known to provide an anti-influenza activity.

Example 6 Activation of TLR-7 by PapMV VLP ssRNA

C57BL/6, TLR7 knockout (KO), MYD88 KO and IRF5/7 KO mice (3-5 mice pergroup) were immunized intravenously (i.v.) with 100 μg PapMV VLP ssRNAor 100 μl PBS. Splenocytes were isolated 24 hours post-immunization andCD86 and CD69 expression in dendritic cells (DCs), CD8⁺ T cells and Bcells was analyzed. Cells were sorted by FACS and the level of CD86 andCD69 was evaluated by fluorescence intensity though the binding of aCD69 or CD86 specific antibody. The results are presented in FIG. 8 as aratio of the Mean Fluorescence Intensity (MFI) of the analyzed sample onthe PBS sample.

In brief, these results show that antigen-presenting cells, such as DCsand B cells and CD8+ T cells, are activated by PapMV VLP ssRNAnanoparticles. Activation is dependent on IRF5/7, Myd88 and TLR-7, asactivation is lost in mice that are knockouts in IRF5/7, Myd88 or TLR7.It is believed that TLR-7 is triggered through the ssRNA that iscontained in the VLPs. Experiments performed with the coat protein ofPapMV (in monomeric or other low molecular weight form) failed toactivate TLR-7.

IRF5/7 are the interferon responsive factors that are induced uponstimulation of TLR-7 and lead to production of interferon alpha. TheMyd88 molecule is an adaptor molecule that is responsible for thetransfer of the signals triggered by TLR-7. The cascade of the reactionis proposed to be: 1) triggering of TLR-7 by the ssRNA in the VLPs, and2) engagement of Myd88 followed by the induction of IRF5/7 that willlead to an increase in interferon alpha production. Finally, interferonalpha will contribute to the immunomodulation effects of the PapMV VLPnanoparticles.

Example 7 Involvement of Plasmacytoid Dendritic Cells in PapMV VLPImmunogenicity

C57BL/6 mice (5 per group) were immunized i.v. with 100 μg PapMV VLPssRNA either with or without prior treatment to deplete BST2+ cells. Fordepletion, C57BL/6 mice were injected i.p. with 500 μg of an anti-BST2antibody (mAb 927) at 4811 and 24 h prior to PapMV VLP ssRNAimmunization. CD69, MHC—I and CD86 expression in isolated splenocyteswas analyzed by FACS at 24 h after PapMV VLP ssRNA immunization.

The results are shown in FIG. 9 and indicate that BST2⁺ cells (mainlyplasmacytoid dendritic cells) are important for the immunogenicity ofPapMV VLP ssRNA nanoparticles in mice. Specifically, it was observedthat in mice in which BST2+ cells were depleted, activation of B cells,CD8+ cells and DCs was lost, suggesting that the activation is goingthrough the plasmacytoid dendritic cells.

Example 8 Stimulation of Interferon-α Production of by PapMV VLPs #1

Two groups of C57BL/6 mice, as well as TLR-7 KO and MYD88 KO mice (4mice per group) were immunized i.v. with 100 μg PapMV VLP ssRNA or 100μl PBS. One group of C57BL/6 mice had first been treated with anti-BST2antibody as described in Example 7. IFN-α production in serum and spleenwas monitored by ELISA (VeriKine™ Mouse Interferon Alpha ELISA Kit; PBLInterferonSource) at either 6, 12, 24 and 48 h post-immunization (FIG.10A) or at 6 h after the immunization (FIG. 10B).

The results are shown in FIG. 10 and indicate that IFN-α productionstimulated by PapMV VLP ssRNA nanoparticles depends on MYD88, ILR7 andBST2⁺ cells.

Example 9 Stimulation of Interferon-α Production of by PapMV VLPs #2

C57BL/6 and IFNAR KO mice (3 mice per group) were immunized i.v. with100 μg PapMV VLP ssRNA or 100 μl PBS. CD86, MHC-I and CD69 expression inB lymphocytes and dendritic cells isolated from the spleens of the mice24 h after immunization was assessed by flow cytometry.

The results are shown in FIGS. 11A and B, and indicate that the type IIFN receptor is necessary for the activation of murine immune cells byPapMV VLP ssRNA nanoparticles. Mice that were knockouts for the type IIFN receptor (IFNAR KO) did not show activation of the immune cells byPapMV VLP ssRNA nanoparticles.

Levels of antibody against PapMV VLP ssRNA in the serum of C57BL/6 andIFNAR KO mice (9 mice per group) at day 4, 8, 12, 20 and 30 afterimmunization with 100 μg PapMV VLP ssRNA were analyzed by indirect ELISAmeasuring total IgG binding to PapMV VLP ssRNA coated plate.

The results are shown in FIG. 11C and indicate that the absence of typeI IFN signalling causes a significant delay in the antibody responseagainst the PapMV VLP ssRNA nanoparticles.

Example 10 Pre-Treatment with PapMV VLPs Helps to Control ChronicInfection

LCMV is a relevant animal model of chronic infection (such as HCVinfection). The clone 13 variant of LCMV establishes a persistentinfection in mice. LCMV infection, like HCV infection, is largelycontrolled by CTLs and exhaustion of the CTL response is associated withPD-1 expression.

C57BL/6 and TLR7 knockout (KO) mice (3-6 mice per group) were treatedi.v. with 100 μg PapMV VLP ssRNA, 100 μg R837 (a commercially availableTLR-7 ligand) or 100 μl PBS 6 hours before infection (i.v.) with 2×10⁶PFU LCMV clone 13. Blood samples were taken at day 5, 11, 15, 25 and 45to evaluate the viral titer by LCMV focus-forming assay. Mice weresacrificed 15 days or 45 days post-infection for analysis of the immuneresponse in the spleen by FACS and of the viral titer in the spleen,liver, kidney and brain by LCMV focus-forming assay on MC57 fibroblastsusing a rat anti-LCMV-NP monoclonal Ab (VL-4) as previously described(Lacasse et al., 2008, J. Virology, 82:785-794). The viral kinetics ofLCMV clone 13 in the blood of the C57BL/6 mice are depicted in FIG. 12and show that pre-treatment with PapMV VLP ssRNA nanoparticles controlchronic infection induced by LCMV.

The viral titers in spleen, kidney, liver and brain of C57BL/6 andTLR7KO mice at day 15 post-infection are shown in FIG. 13 anddemonstrate that pre-treatment with PapMV VLP ssRNA nanoparticlesdecreases the viral load in different organs with greater efficiencythan a commercial TLR7 ligand (R837) and in a TLR7 dependent manner. Itis believed that the TLR-7 ligand in the PapMV VLP ssRNA nanoparticicsis the ssRNA component, which represents approximately 5% of themolecule. As such, although 100 μg of each was administered to the mice,the PapMV VLP ssRNA nanoparticles are more than 20-fold more effectivethan R837 in reducing the LCMV viral load in the mice.

FIG. 14 shows that administration of PapMV VLP ssRNA nanoparticlesbefore infection with LCMV clone 13 increases the functionality of GP33specific CD8⁺ T cells. Similar results were obtained for NP396 specificCD8⁺ T cells. In particular, FIG. 14F shows that the amount of PD-1expressed in GP33 specific CD8⁺ T lymphocytes is significantly decreasedby pre-treatment with PapMV VLP ssRNA nanoparticles. PD-1 is anindicator of immune exhaustion and its expression is a characteristic ofLCMV clone 13 infection. Pre-treatment of the mice with PapMV VLP ssRNAnanoparticles resulted in the PD-1 level remaining as low as in theuninfected mice suggesting that the immune system is not exhausted inthese mice, which is why they are able to resist infection.

FIG. 15 shows the viral titers in spleen, kidney, liver and brain ofC57BL/6 mice at day 45 post-infection. This result indicates that thedecrease in viral load resulting from pre-treatment with PapMV VLP ssRNAnanoparticles is still evident several weeks after treatment.

Example 11 Activation of Human Monocytes In Vitro by PapMV VLPs

Human PBMCs were isolated by Ficoll gradient and treated with 100 μg/mlPapMV VLP ssRNA or PBS. At 18 h post-treatment, CD14⁺CD11b⁺ cellpopulation (monocytes) were analyzed for CD86 expression by flowcytometry.

The results are shown in FIG. 16 and indicate that human monocytes arealso activated by PapMV VLP ssRNA nanoparticles. These results arerepresentative of three independent experiments.

Example 12 Induction of an Anti-Bacterial Response by PapMV VLPs

Mice, 10 per group, were treated twice at 7-day intervals via theintranasal route with buffer alone (10 mM Tris pH8) or with 60 μg ofPapMV VLP ssRNA. At day 3 post-treatment, the mice were infected with220 CFU (colony forming units) of a virulent Streptococcus pneumoniaestrain.

Survival was monitored closely every 12 hours over 4 days. The resultsare shown in FIG. 17. All mice in the group treated with PapMV VLP ssRNAnanoparticles survived the infection. The group treated with the buffershowed 70% survival.

Although the dose of Streptococcus pneumoniae used in this Example was asub-lethal dose, the data strongly suggests that pre-treatment withPapMV nanoparticles will provide protection against a bacterialinfection through the induction of an innate immune response in thelungs. This example and the preceding examples demonstrate that theprotection conferred by the PapMV nanoparticles is non-specific as it iseffective against infection with viruses and bacteria.

Example 13 Treatment of LCMV Chronic Infection Using PapMV VLPs

C57BL/6 Mice (3 per group) were infected i.v. at day 0 with 2×10⁶ PFULCMV clone 13 and treated i.v. once/day with 100 μg PapMV VLP ssRNA or100 μl PBS either at days 1, 2, 3, 4 and 5 (Group A), or at days 6 and 7only (Group B). Blood samples were taken at day 5, 10 and 15 and micewere sacrificed at day 15 post-infection for analysis of the viral titerby LCMV focus-forming assay in blood, spleen, kidney and brain.

Viral titers found in the blood of the animals are shown in FIG. 18.Although at day 15, mice treated with PapMV VLP ssRNA nanoparticlesshowed the same titers as the controls, a significant reduction of viraltiters was observed at day 10 in both groups of mice (close to a log 10reduction in the animals of Group A). This result strongly suggeststhat, with adjustment to the treatment regimen, further decreases inviral load in mice treated with PapMV VLP ssRNA nanoparticles will beachievable. For example, the number of treatments could be increased. Astreatments provided at days 1 to 5 or 6 and 7 showed a decrease in LCMVtiters, it is likely that an increase in the number of treatments afterdays 6 and 7 will provide a further decrease in viral load.Alternatively, or in addition, the amount of PapMV VLPs administeredcould be increased, for example, to 200 μg per dose.

Viral titers found in various organs of the animals are shown in FIG.19. While the viral load in the brain of animals treated at days 6 and 7(Group B) showed a significant reduction, viral loads in other organs ofthe treated mice did not show a significant reduction. This is mostlikely because the titers were measured at day 15, which allowed theinfection sufficient time to ‘kick back’ after treatment. Subsequenttreatments to days 6 and 7 would be anticipated to lead to a moresignificant decrease in viral loads.

Example 14 Multiple Treatments with PapMV VLPs Prolong the ProtectionPeriod

Example 2 demonstrates that the protection induced by the treatment withPapMV VLPs appears to persist for a period of about 5 days. Toinvestigate if treatment with multiple doses of PapMV VLPs could providea longer period of protection, mice were treated following the generalregimen described in Example 1 with PapMV VLPs containing ssRNA once(1×), twice (2×), 5 times (5×) or 10 times (10×) at 1-week intervals.Three days after the final treatment, the mice were challenged withinfluenza WSN/33 virus as described in Example 2.

The weight loss of the mice is shown in FIG. 20. It was also noted thatthe animals were not affected by the multiple treatments and gainedweight normally during the treatment period in line with the controlanimals.

These results show that multiple treatments can extend the period ofprotection induced by the PapMV VLP nanoparticles to more than 10 weeks.The results also demonstrate that multiple treatments with PapMV VLPs donot exhaust the innate immunity of the animal. Finally, as it is knownthat antibodies to the PapMV VLPs appear 7 days after the firsttreatment and increase with the booster treatments, these resultsdemonstrate that the ability of the PapMV VLPs to trigger the innateimmune response is not impacted by the production of antibodies.

Example 15 Induction of Neutrophil Recruitment by PapMV VLPs

Mice were submitted to 2 instillations of PapMV VLPs containing ssRNAaccording to the protocol of Example 1 and broncho-alveolar lavage (BAL)was performed 6 hours after the second treatment. The results are shownin FIG. 21. Neutrophils found into the BAL of mice treated with PapMVVLPs are circled in FIG. 21B. Three times more neutrophils were observedin the treated mice compared to the control group.

Neutrophils represent the first line of defense. This Exampledemonstrates that neutrophils are recruited rapidly in mice treated withPapMV VLPs; just 6 hours after treatment. Neutrophils are known to playa key role in the control of bacterial and viral infection in the lungsand thus likely play a role in the protection observed in PapMV treatedmice.

Example 16 Induction of a Mucosal Immune Response by PapMV VLPs

Balb/C mice (10 per group) were treated with two instillations of 20 μgPapMV VLP ssRNA combined with 2 μg of the trivalent inactivated fluvaccine (TIV) at 14 day intervals. Bleedings were performed at day 0, 14and 28. Following the same protocol, another group of mice wereimmunized animals by the s.c. route for comparison. Mice were challengedat day 15 with 1LD₅₀ of the influenza WSN/33 virus and weight loss wasfollowed over a 14 day period.

IgG titers were measured in the blood of the immunized animals by ELISAusing antibodies to the TIV and the results are shown in FIG. 22. Theaddition of PapMV VLPs to the TIV increased significantly the total IgGand the IgG2a response as compared with the group immunized with TIValone when the same route of immunization was used. Interestingly, thes.c. route was more efficient than the i.n. route for production oftotal IgG and IgG2a in the blood of the animal.

Antibody titers were measured in the broncho-alveolar lavage (BAL) andin the faeces of the immunized animals by ELISA using antibodies to theTIV and the results are shown in FIG. 23. From these results, it isclear that only i.n. treatment triggers production of IgA in the BAL.The addition of PapMV VLPs to the TIV increased significantly the amountof IgA in the lungs as compared to instillation with TIV alone.Significantly higher total IgG in the BAL was also observed in theanimals treated with PapMV VLPs in combination with the TIV, as comparedto the TIV alone group. The amount of total IgG in the BAL obtained frommice treated intranasally with PapMV VLPs in combination with the TIVadministered by i.n. was not significantly different from that in micetreated subcutaneously with the combination. Finally, it was interestingto note that a mucosal immune response was also observed in theintestines of the mice treated intranasally with the combination asshown by the presence of IgA directed to TIV in this organ (FIG. 23C).

Weight loss in the mice after challenge with the influenza virus isshown in FIG. 24. The challenge revealed that immunization by theintranasal route is more robust and efficient in protecting mice to aheterosubtypic strain than immunization by the s.c. route. In the groupimmunized by the i.n. route with the combination of PapMV VLPs and TIV,the mice gained weight and did not show any symptoms. The combinationadministered s.c. provided only a partial protection. Completeprotection can, however, be achieved using s.c. administration of 3 μgof TIV with 30 μg of PapMV VLPs. All the other groups that wereimmunized with TIV alone (by either route), PapMV VLPs alone or thecontrol buffer were not protected, showed symptoms of disease and lostsignificant amounts of weight.

The results from this experiment demonstrate that PapMV VLPs can act asa mucosal adjuvant. The ability of an adjuvant to trigger a mucosalimmune response is important for effective prevention or treatment ofinfections and diseases caused by micro-organisms that gain access tothe body via mucosal membranes, including influenza, tuberculosis, andH. pylori infections. The presence of IgG in the faeces of the immunizedanimals suggests that i.n. vaccinations using PapMV VLPs as adjuvantcould be used to protect against bacterial or viral infection in theintestine. In addition, since the mucosal immune response triggered bythe PapMV VLPs is general, i.n. vaccinations using PapMV VLPs asadjuvant could potentially also be used to protect against bacterial orviral infection (such as HIV-1) in the vaginal mucosa.

Although in this experiment no protection was seen in mice treated i.n.with PapMV VLPs alone, this is consistent with the results in theprevious examples which indicate that the non-specific protectioninduced by PapMV VLPs lasts only for a period of about 5 days. In thisexperiment, the challenge was performed 14 days after the secondinstillation of VLPs.

Example 17 Activation of TLR-2 and CD14 by PapMV VLPs

As demonstrated in the preceding Examples, PapMV VLPs prepared inbacterial host cells and PapMV VLPs prepared by in vitro self-assemblywith ssRNA are both able to stimulate the innate immune response.However, VLPs prepared by the two different methods, activate differentTLRs. As shown above, PapMV VLPs prepared by in vitro self-assembly withssRNA activate TLR-7. In contrast, PapMV VLPs prepared by expression ofthe PapMV coat protein and self-assembly in E. coli cells as previouslydescribed (Tremblay et al., 2006, ibid.), activate TLR-2 and CD14.

In brief, THP1-XBlue™-CD14 cells (InvivoGen, San Diego, Calif.) weretreated with 100 μg PapMV VLPs (prepared according to Tremblay et al.)or a known TLR ligand (100 μg lipotcichoic acid from S. aureus (LTA):TLR2 and CD14 ligand; 1 μg Pam3SCK4: TLR2 ligand; or 10 μg flagellin:TLR5 ligand) and either an anti-CD14, anti-TLR2 or anti-TLR5 antibody.THP1-XBlue™-CD14 cells harbour several TLRs (including TLR2, 4, 5) andhave been modified to produce a blue colour when a TLR is engaged with aligand. Upon engagement, the cells become blue and the strength of theengagement can be readily evaluated using a spectrophotometer.Measurements were made after a 24 hour incubation of the cells at 37° C.

The results are shown in FIG. 26. Antibodies (Ac) directed to CD14, TLR2or TLR5 blocked engagement of the respective TLR or CD14 and revealedwhat interactions were being made by each test molecule. A significantdecrease in optical density was observed when the anti-TLR2 antibody wasused with the PapMV VLPs, and a strong decrease observed when theanti-CD 14 antibody was used. In this experiment, the antibody to TLR2did not work as well as expected as a higher decrease should have beenobserved when Pam3CSK4 (a known TLR2 ligand) was used. It is likely theamount of Pam3CSK4 used in the experiment was too high.

The difference in TLR activation seen with the PapMV VLPs assembled inbacteria may be due to the detergent treatment that the VLPs undergoafter isolation from the bacterial cells. This treatment may affect thesurface of the PapMV VLPs, for example to expose hydrophobic residues,and result in the VLPs becoming a ligand of TLR2. In contrast to TLR7,which is present in the endosome, both TLR2 or CD14 are surface exposedon immune cells.

Example 18 Method for Preparing PapMV VLPs Comprising ssRNA

This example describes a process for preparing PapMV VLPs in vitro byassembling recombinant PapMV Coat Protein (rCP) onto synthetic RNAtemplates (SRT) to produce recombinant VLPs (rVLPs). The rVLPs are rodshaped nanoparticles 15 nm wide, and 50 to thousands nm in length. Theprocess is summarized in the flow chart presented in FIG. 27.

Production of rCP

rCP harbouring a 6×His-tag was produced in E. coli transformed withplasmid DNA containing the rCP gene under the control of an induciblepromoter. In brief, the PapMV CP was cloned into the pQE80 vector(QIAGEN) flanked by the restriction enzyme NcoI and BamHI and theprotein was expressed under the control of the T5 promoter. E. coliBD-792 was used for expression. Transformed bacteria were grown instandard culture medium. Protein expression was triggered by addition ofa biochemical inducer to the culture medium (0.7-1 mM IPTG for 6-9 h at22-25° C.). At the end of the induction period, cells were harvested,suspended in lysis buffer (10 mM Tris pH 8.0, 500 mM NaCl) and rupturedmechanically using a French press, homogenizer or sonicator. Cell lysatewas clarified by removal of genomic DNA by standard DNase treatment andremoval of large cell fragments and membranes by centrifugation ortangential flow filtration (300 kDa to 0.45 μm MWCO membranes). rCP wascaptured on an ion-matrix affinity resin and eluted with a pH gradientor imidazole using standard procedures. The rCP was subsequentlypurified from endotoxins by anion exchange chromatography/filtration andfrom small low MW molecules by tangential flow filtration (0 to 30 kDaMWCO membranes). Any contaminating imidazole present in the rCP solutionwas removed by dialysis or tangential flow filtration (5 to 30 kDa MWCOmembranes). The final rCP protein solution was sterilized by filtration.The sterile product can be stored at 2-8° C. and is stable for severalyears.

Production of SRT

The sequence of the SRT is provided in FIG. 28 [SEQ ID NO:5]. The SRT isbased on the genome of PapMV and harbours the PapMV coat proteinnucleation signal at the 5′-end (boxed in FIG. 28). The remainingnucleotide sequence is poly-mutated in that all ATG codons have beenmutated for TAA stop codons. The first 16 nucleotides of the sequence(underlined in FIG. 28) comprise the T7 transcription start site locatedwithin the pBluescript expression vector and are present within the RNAtranscript. Pentameric repeats are underlined in FIG. 28. The entiretranscript is 1522 nucleotides in length.

DNA corresponding to the SRT was inserted into a DNA plasmid including aprokaryotic RNA polymerase promoter using standard procedures. Therecombinant plasmid was used to transform E. coli cells and thetransformed bacteria were subsequently grown in standard culture medium.The plasmid DNA was recovered and purified from the cell culture bystandard techniques, then linearized by cleavage with a restrictionenzyme (MluI) at the point in the DNA sequence immediately after thelast nucleotide that was to be present in SRT.

Transcription of SRT was conducted with T7 RNA polymerase using theRiboMAX™ kit (Promega, USA) following the manufacturer's recommendedprotocol. The expression vector was designed such that transcriptsoriginating from the RNA polymerase promoter were released from the DNAtemplate at the DNA point of cleavage. The SRT was purified to removeDNA, protein and free nucleotides by tangential flow filtration using a100 kDa MWCO membrane. The final RNA solution was sterilized byfiltration. The sterile product can be stored below −60° C. and isstable for several years.

Production of rVLPs

rVLPs were assembled in vitro by combining the rCP and SRT. The assemblyreaction was conducted in a neutral buffered solution (10 mM Tris-HCl pH8). The assembly reaction was conducted using a protein:RNA ratiobetween 15-30 mg of protein for 1 mg RNA. The newly assembled rVLPs wereincubated with a low amount of RNase (0.0001 μg RNAse per μg RNA) toremove any RNA protruding from the rVLPs. This step improves thesolubility of the rVLPs. The blunted-rVLPs were then purified fromcontaminants and free rCP (unassembled monomeric rCP) by diafiltrationusing 10-100 kDa MWCO membranes. The final rVLP liquid suspension wassterilized by filtration. The sterile product can be stored at 2-8° C.and is stable for several years.

The disclosure of all patents, publications, including published patentapplications, and database entries referenced in this specification areexpressly incorporated by reference in their entirety to the same extentas if each such individual patent, publication, and database entry wereexpressly and individually indicated to be incorporated by reference.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention. All such modifications as would be apparent to oneskilled in the art are intended to be included within the scope of thefollowing claims.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled) 20.(canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled) 29.(canceled)
 30. (canceled)
 31. (canceled)
 32. A method of stimulating aninnate immune response in a subject comprising administering to thesubject an effective amount of a composition comprising papaya mosaicvirus (PapMV) or PapMV virus-like particles (VLPs), thereby preventing,or decreasing the severity of, a microbial infection in the subject. 33.The method according to claim 32, wherein the microbial infection is aninfection with a microorganism that gains access to the subject's bodyvia mucosal membranes.
 34. The method according to claim 32, wherein themicrobial infection is a viral infection.
 35. The method according toclaim 32, wherein the microbial infection is a bacterial infection. 36.The method according to claim 32, for preventing a microbial infectionin the subject.
 37. The method according to claim 32, for decreasing theseverity of a microbial infection in the subject.
 38. The methodaccording to claim 32, wherein the immune response is stimulated at amucosal surface.
 39. The method according to claim 38, wherein thecomposition is administered in combination with one or more antigens.40. The method according to claim 32, wherein the composition isadministered via an intranasal route.
 41. The method according to claim32, wherein the composition comprises PapMV.
 42. The method according toclaim 32, wherein the composition comprises PapMV VLPs.
 43. The methodaccording to claim 42, wherein the PapMV VLPs comprise ssRNA.
 44. Amethod of protecting a subject against infection with a pathogencomprising administering to the subject an effective amount of acomposition comprising papaya mosaic virus (PapMV) or PapMV virus-likeparticles (VLPs), wherein the composition stimulates the innate immuneresponse in the subject.
 45. The method according to claim 44, whereinthe pathogen is a microorganism that gains access to the subject's bodyvia mucosal membranes.
 46. The method according to claim 44, wherein thepathogen is a virus.
 47. The method according to claim 46, wherein thevirus is an influenza virus, flavivirus, parainfluenza virus,respiratory syncytial virus, coronavirus, adenovirus or rhinovirus. 48.The method according to claim 44, wherein the pathogen is a bacterium.49. The method according to claim 48, wherein the bacterium is Bacillusanthracis, Yersinia pestis, Francisella tularensis, Streptococcuspnemoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderiacepacia, Corynebacterium diphtheriae, Legionella pneumophila, Mycoplasmapneumoniae, Chlamydophila pneumoniae, Mycobacterium tuberculosis,Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae,Escherichia coli or Coxiella burnetii.
 50. The method according to claim44, wherein the composition is administered to the subject between 4hours and 6 days prior to expected exposure of the subject to thepathogen.
 51. The method according to claim 44, wherein the compositionis administered to the subject in multiple doses.
 52. The methodaccording to claim 44, wherein the subject is immunocompromised,undergoing chemotherapy or taking immunosuppressive drugs.
 53. Themethod according to claim 44, wherein the composition prevents immuneexhaustion in the subject.
 54. The method according to claim 44, whereinthe composition is administered via an intranasal route.
 55. The methodaccording to claim 44, wherein the composition comprises PapMV.
 56. Themethod according to claim 44, wherein the composition comprises PapMVVLPs.
 57. The method according to claim 56, wherein the PapMV VLPscomprise ssRNA.
 58. A method of treating a chronic or recurrentmicrobial infection comprising administering to a subject having achronic or recurrent microbial infection a composition comprising PapayaMosaic Virus (PapMV) or PapMV virus-like particles (VLPs).
 59. Themethod according to claim 58, wherein the composition is administered incombination with one or more conventional therapies.
 60. The methodaccording to claim 58, wherein the microbial infection is a bacterialinfection.
 61. The method according to claim 58, wherein the microbialinfection is a viral infection.
 62. The method according to claim 61,wherein the viral infection is a hepatitis C virus infection or humanimmunodeficiency virus infection.
 63. The method according to claim 61,wherein the composition decreases viral load in the subject.
 64. Themethod according to claim 58, wherein the composition is administeredvia an intranasal route.
 65. The method according to claim 58, whereinthe composition comprises PapMV.
 66. The method according to claim 58,wherein the composition comprises PapMV VLPs.
 67. The method accordingto claim 66, wherein the PapMV VLPs comprise ssRNA.
 68. A kit comprisinga container having contained therein a pharmaceutical compositioncomprising papaya mosaic virus (PapMV) or PapMV virus-like particles(VLPs), the container adapted to deliver the pharmaceutical compositionby an intranasal, pulmonary or vaginal route.
 69. The kit according toclaim 68, wherein the container is an inhaler, nebulizer or nasal spraydevice.